01 Fastdigest Conventional Restriction Enzymes

FastDigest and Conventional Restriction Enzymes...................................................
All Fermentas products are manufactured in class D clean room facilities, qualified and certified as per EU directives and ISPE guidelines. Fermentas quality assurance is carried out according to ISO9001 quality and ISO14001 environmental management systems, guaranteeing batch-to-batch reproducibility. Integration of our clean room and ISO systems ensures stability and the absence of contaminants in all of our products.
PureExtreme Quality. ..................................................................................................... 2 Labeled Oligonucleotide (LO) Test.................................................................................. 2 Navigation Guide. ............................................................................................................. 3 Description of Icons......................................................................................................... 4 FastDigest Restriction Enzymes. ................................................................................... 5 Activity and Quality Control Assays................................................................................ 6 Storage and Shipping....................................................................................................... 6 Product List....................................................................................................................... 7 Product Description. ...................................................................................................... 11 Protocols and Recommendations................................................................................. 70 1.1. Fast DNA digestion. ................................................................................................ 70 1.2. Reaction set-up for digestion of multiple DNA samples. ........................................... 70 1.3. Double and multiple digestion of DNA . .................................................................. 70 1.4. Scaling up DNA digestion reaction.......................................................................... 70 Reaction Conditions....................................................................................................... 71 Conventional Restriction Enzymes................................................................................ 75 Activity and Quality Control Assays.............................................................................. 75 Storage and Shipping..................................................................................................... 75 Product List and Cross-reference to FastDigest Restriction Enzymes.................... 76 Product Description. ...................................................................................................... 80 Nicking Enzymes........................................................................................................ 157 Homing Enzyme. ......................................................................................................... 159 Protocols and Recommendations............................................................................... 160 1.5. DNA digestion..................................................................................................... 160 1.6. Digestion of PCR products................................................................................... 160 1.7. Setting up double digestion.................................................................................. 160 1.8. Stability during prolonged incubation.................................................................... 160 1.9. Dilution of restriction enzymes. ............................................................................. 160 1.10. Partial digestion of DNA..................................................................................... 160 1.11. Digestion of agarose-embedded DNA. ................................................................. 161 1.12. Inactivation of restriction enzymes...................................................................... 161 Reaction Conditions .................................................................................................... 162 Reaction Buffers......................................................................................................... 162 Recommended Reaction Conditions............................................................................ 163 Activity of Mesophilic and Thermophilic Enzymes at 37C............................................ 167 Double Digestion in Universal Tango Buffer............................................................... 168 Digestion of Agarose-Embedded DNA......................................................................... 170 Cleavage Efficiency Close to the Termini of PCR Fragments.......................................... 171 Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site................................................................................................................ 172 Common Properties. ....................................................................................................... 173 Classification of Restriction Enzymes........................................................................ 173 Site Preferences by Restriction Endonucleases........................................................ 173 Star Activity...................................................................................................................174 Digestion of Methylated DNA....................................................................................... 175 Effect of Dam Methylation on DNA Cleavage................................................................ 176 Effect of Dcm Methylation on DNA Cleavage................................................................ 177 Effect of CpG Methylation on DNA Cleavage................................................................ 178 Effect of EcoKI and EcoBI Methylation on DNA Cleavage.............................................. 181 Newly Generated Cleavage Sites................................................................................. 182 Recognition Sites Resulting from Ligation of Blunt DNA Ends. ....................................... 182 Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends. ............. 192 Recognition Sites Resulting from Fill-in of 5-overhang and Self-ligation....................... 202 Recognition Sites Resulting from Removal of 3-overhang and Self-ligation................... 206 Selection Guides.............................................................................................................. 207 Alphabetic List of Commercially Available Restriction Enzymes............................. 207 Alphabetic List of Recognition Sequences................................................................. 219 Enzyme Recognizing 2 bp Long Targets..................................................................... 222 Alphabetic List of Enzymes Recognizing 4 bp Long Targets. ................................... 222 Alphabetic List of Enzymes Recognizing 5 bp Long Targets. ................................... 222 Alphabetic List of Enzymes Recognizing 6 bp Long Targets. ................................... 223 Alphabetic List of Enzymes Recognizing 7 bp Long Targets. ................................... 224 Alphabetic List of Enzymes Recognizing 8 bp Long Targets. ................................... 224 Commercial Restriction Enzymes Generating 5-protruding Ends........................... 225 Commercial Restriction Enzymes Generating 3-protruding Ends........................... 228 Commercial Restriction Enzymes Generating Blunt Ends. ....................................... 230 Commercial Restriction Enzymes Cleaving DNA on the Both Sides of their Recognition Sequence. ................................................................................................ 230 Troubleshooting Guide.................................................................................................... 231
FastDigest & CONVENTIONAL RESTRICTION ENZYMES
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1. FastDigest & CONVENTIONAL RESTRICTION ENZYMES
FastDigest and Conventional Restriction Enzymes

Restriction enzymes recognize specific nucleotide sequences and cleave DNA molecules at a position either within or outside their recognition site. These enzymes are important tools for numerous applications, including studies of DNA primary structure and recombinant DNA technology. The best studied and the most widely used are type II restriction endonucleases. More than 3800 type II restriction enzymes, exhibiting almost 290 different specificities, have been isolated. Since 1977 Fermentas has discovered approximately 30% of all known restriction enzymes. We are a leading global enzyme manufacturer, supplying both conventional restriction enzymes and innovative restriction enzyme line for rapid DNA digestion in one universal buffer FastDigest restriction enzymes. Fermentas offers 176 FastDigest and 201 conventional restriction enzymes.
PureExtreme Quality
Fermentas Restriction enzymes are produced in clean room class D facilities under the ISO 9001:2008 quality management system and are subjected to extensive quality control. Fermentas restriction enzymes pass all industry standard quality control assays, as well as the unique Labeled Oligonucleotide (LO) Test, which is the most sensitive test for the detection of trace activities of endodeoxyribonucleases, exodeoxyribonucleases and phosphatases see Fig. 1.1. A warranty is assigned and an expiry date is listed both on the product label and in the Certificate of Analysis supplied with each product. Product lots are monitored to meet the quality specifications up to the expiry date.
Labeled Oligonucleotide (LO) Test

The Labeled Oligonucleotide Test was designed at Fermentas for detection of trace contaminating activities in enzyme, nucleotide and reagent solutions. This unique test allows us to detect trace activities of endo-, exodeoxyribonucleases and phosphatases in enzyme preparations. Therefore we are able to bring to the market only highest quality and performance products. Single-stranded and double-stranded 5-[32P]-labeled synthetic oligonucleotides containing no recognition sites for restriction enzymes are used as substrates for the LO test. The labeled oligonucleotides are incubated with excess of restriction enzyme, separated on a polyacrylamide gel under denaturing conditions and analyzed by phospho-imaging. The presence of contaminating endo- and exodeoxyribonucleases results in the degradation of the labeled substrates (see Fig. 1.1), while decreased specific radioactivity indicates the presence of contaminant phosphatases. The restriction enzyme passes this quality control test if there is neither degradation of labeled oligonucleotides nor a decrease in specific radioactivity.
Labeled Oligonucleotide Test

Radiolabeled ss and ds oligonucleotides mixed with an excess of enzyme Incubated at 37C for 4hours Denatured and separated by PAGE Band pattern imaged and analyzed Patterns Typical for:
Control Pure enzyme Contaminated enzyme
ss ds
ss ds
ss ds
Degradation due to contamination with non-specific nucleases
Figure 1.1. Labeled Oligonucleotide (LO) Test. ss single-stranded radiolabeled oligonucleotide ds double-stranded radiolabeled oligonucleotide Pure enzyme Fermentas NotI Contaminated enzyme competitors NotI
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Navigation Guide
Table 1.1. FastDigest and conventional restriction enzymes.
& CONVENTIONAL RESTRICTION ENZYMES
To find or select a restriction enzyme Table 1.2. FastDigest restriction enzymes Table 1.4. Fermentas conventional restriction enzymes By name Table 1.25. Commercially available restriction enzymes On-line. REsearch at www.fermentas.com/research Table 1.26. Recognition sequences of restriction enzymes Table 1.27. Enzyme recognizing 2 bp long targets Table 1.28. Enzymes recognizing 4 bp long targets Table 1.29. Enzymes recognizing 5 bp long targets By recognition sequence Table 1.30. Enzymes recognizing 6 bp long targets Table 1.31. Enzymes recognizing 7 bp long targets Table 1.32. Enzymes recognizing 8 bp long targets On-line. REsearch at www.fermentas.com/research Table 14.15. Number of recognition sites in DNA molecules By number of recognition sites Appendix. Phage and plasmid DNA. in DNA On-line. REviewer at www.fermentas.com/reviewer Table 1.33. Commercial restriction enzymes generating 5-protruding ends Table 1.34. Commercial restriction enzymes generating 3-protruding ends By type of generated DNA ends Table 1.35. Commercial restriction enzymes generating blunt ends Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence On-line. REsearch at www.fermentas.com/research Table 1.3. Reaction conditions for FastDigest restriction enzymes By cleavage efficiency close to Table 1.10. Cleavage efficiency close to the termini of PCR fragments termini of DNA Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site Table 1.12. Fermentas isoschizomers and neoschizomers with differing sensitivities to target methylation Tables 1.13, 1.14. Effect of Dam methylation By sensitivity to DNA methylation Tables 1.15, 1.16. Effect of Dcm methylation Tables 1.17, 1.18. Effect of CpG methylation Tables 1.19, 1.20. Effect of EcoBI and EcoKI methylation Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends By newly generated recognition sites Table 1.23. Newly generated recognition sites resulting from fill-in of 5-overhang and self-ligation Table 1.24. Newly generated recognition sites resulting from removal of 3-overhang and self-ligation To perform fast digestion of DNA in universal FastDigest buffer Fast DNA digestion reaction set-up Protocols and recommendations for fast DNA digestion Digestion of different DNA substrates Digestion close to DNA termini Table 1.3. Reaction conditions for FastDigest restriction enzymes Inactivation of enzyme after digestion Incubation time without star activity Compatibility of FastDigest enzyme buffer with downstream 2.1. Activity of DNA/RNA modifying enzymes in Fermentas buffers applications Troubleshooting Table 1.37. Troubleshooting guide for DNA digestion To perform DNA digestion with conventional restriction enzymes DNA digestion reaction set-up Protocols and recommendations for DNA digestion with conventional restriction enzymes On-line. DoubleDigest at www.fermentas.com/doubledigest Double/multiple digestion of DNA Table 1.8. Double digestion in universal Tango buffer Table 1.6. Reaction conditions for restriction enzymes Reaction conditions for individual enzymes Digestion close to DNA termini Digestion of agarose-embeded DNA Inactivation of enzyme after digestion How to identify and avoid star activity Compatibility of conventional restriction enzyme buffers with downstream applications Partial digestion of DNA How to dilute restriction enzymes Stability during prolonged incubation Enzyme activity at non-optimal temperature Troubleshooting guide Table 1.6. Reaction conditions for restriction enzymes Table 1.5. Reaction buffers for restriction enzymes Table 1.10. Cleavage efficiency close to the termini of PCR fragments Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 1.11 Table 1.9. Digestion of agarose-embedded DNA 1.12. Inactivation of restriction enzymes Table 1.6. Reaction conditions for restriction enzymes Description of star activity of restriction enzymes 2.1. Activity of DNA/RNA modifying enzymes in Fermentas buffers 1.10. Partial digestion of DNA 1.9. Dilution of restriction enzymes Table 1.6. Reaction conditions for restriction enzymes Table 1.7. Activity of mesophilic and thermophilic enzymes at 37C Table 1.37. Troubleshooting guide for DNA digestion
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274 232 160 168 163 163 162 171 172 161 161 163 174 274 160 160 163 167 232
For patent and license information see p.557
Bulk quantities and custom formulations available upon request
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Description of Icons
FastDigest Enzyme. Enzyme is available in FastDigest format (see p.5). Buffers. Letters in the buffer icon indicate the recommended buffer for a specific restriction enzyme. FastDigest and FastDigest Green Buffers are indicated by F icon. B (blue), G (green), O (orange), R (red) and Tango (yellow) correspond to the color codes of the Five Buffer System for conventional restriction enzymes. The Unique icon indicates conventional restriction enzymes that require a special buffer, which is supplied with the enzyme (see p.162). Additives. Indicates additives required to obtain the stated enzyme activity. Solutions of S-adenosylmethionine and oligonucleotides are supplied with enzymes. DTT (#R0861) is available separately (p.476). Incubation Temperature. Indicates the optimal incubation temperature in degree Celsius (C). Ligation Efficiency. Indicates the ligation efficiency of DNA fragments generated by digestion with the restriction enzyme (see p.6 and p.75). Star Activity. Indicates restriction enzymes prone to star activity (see p.174). Sensitivity to Methylation. DNA cleavage by the restriction enzyme is blocked or impaired by Dam, Dcm or CpG methylation within the target sequences (see p.175). Sensitivity to Overlapping Dam, Dcm or CpG Methylation. The target site may be methylated in certain sequence contexts (overlapping methylation). This will result in blocked or impaired DNA cleavage (see p.175). Thermal Inactivation. Indicates conditions for thermal inactivation of the enzyme (seep.161). Indicates that only small amounts of restriction enzyme (up to 10 units) can be thermally inactivated. High Concentration. Indicates enzymes available at a high concentration (50u/l). Genome Qualified. Indicates that the restriction enzyme cleaves agarose-embedded DNA (see p.161). Recombinant Enzyme. Indicates enzymes purified from recombinant E.coli. Blue/White Certified. Enzyme was tested by the Blue/White cloning assay (see p.6 and p.75). LO Certified. Enzyme was tested by the labeled oligonucleotide (LO) assay (see p.2 and p.75).
Single letter code

R = G or A; Y = C or T; W = A or T; M = A or C; K = G or T; S = C or G; H = A, C or T; V = A, C or G; B = C, G or T; D = A, G or T; N = G, A, T or C.
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RESTRICTION ENZYMES
FastDigest Restriction Enzymes

Description
FastDigest enzymes are an advanced line of restriction enzymes for rapid DNA digestion. All FastDigest enzymes are 100% active in the universal FastDigest and FastDigest Green buffers and are able to digest DNA in 5-15 minutes. This enables any combination of restriction enzymes to work simultaneously in one reaction tube and eliminates the need for sequential digestions. As an added convenience, the FastDigest Green Buffer allows for direct loading of the reaction mixture on a gel. FastDigest enzymes can be used to digest plasmid, genomic and viral DNA as well as PCR products and do not show star activity even in prolonged incubations. The FastDigest line is ideal for use in applications that require high purity reaction components, performance reliability and simple reaction set-up.
Universal FastDigest Buffer

Two versions of the universal FastDigest buffer are supplied with each enzyme: 10XFastDigest Buffer and 10XFastDigest Green Buffer. All FastDigest enzymes are 100% active in both FastDigest and FastDigest Green buffers. Enzymes used in common downstream applications such as ligation, blunting and dephosphorylation also have 100% activity in both buffers. As an added convenience, the FastDigest Green Buffer contains a density reagent and two tracking dyes that allow for direct loading of the reaction mixture on gels. The blue dye migrates with 3-5 kb DNA fragments in 1% agarose gel and has an excitation peak at 424nm while the yellow dye migrates faster than 10bp DNA fragments in 1% agarose gel and has an excitation peak at 615nm. The presence of the dyes in the FastDigest Green buffer does not interfere with DNA digestion or with downstream applications but may hinder digestion product analysis by fluorescence excitation. For such applications we recommend use of the colorless FastDigest Buffer.
Features
100% activity of all FastDigest enzymes in the new green universal digestion buffer. 100% buffer compatibility with all downstream applications. Complete digestion in 5-15 minutes. Direct loading of reaction mixture on gels.
Applications
Fast clone analysis. Fast preparation of DNA for cloning. Digestion of PCR products. Fast RFLP genotyping. Digestion of difficult-to-cleave DNA.
FastDigest Buffer
FastDigest Green Buffer
M C 1 2 C 3 4 M Figure 1.2. Five minute triple digestion followed by ligation of plasmid DNA in the universal FastDigest and FastDigest Green buffers. M GeneRuler Express DNA Ladder (#SM1551). C undigested plasmid DNA control. 1 plasmid DNA triple digested with FastDigest EcoRI, FastDigest KpnI and FastDigest SmaI in FastDigest Buffer. 2 the reaction mixture after ligation in FastDigest Buffer. 3 plasmid triple digested with FastDigest EcoRI, FastDigest KpnI and FastDigest SmaI in FastDigest Green Buffer. 4 the reaction mixture after ligation in FastDigest Green Buffer.
Figure 1.3. FastDigest Green Buffer. 1 Reaction mixture containing FastDigest Green Buffer before electrophoresis. 2 Reaction mixture containing FastDigest Green Buffer after electrophoresis.
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1. FastDigest RESTRICTION ENZYMES
Activity and Quality Control Assays

Activity Assay
1l of FastDigest enzyme is formulated to cleave 1g of substrate DNA in 5 or 15min at recommended temperature in 1XFastDigest buffer. 1l of FastDigest restriction endonuclease corresponds to 1 FastDigest unit (FDU) of enzyme. In general, enzymes are assayed with phage DNA at 37C. However, some exceptions apply: Restriction enzymes that show optimum activity at temperatures other than 37C are assayed under their optimal temperature. FastDigest enzymes that do not have recognition sites on DNA are assayed with other specific DNA substrates, as indicated in the Certificates of Analysis. FastDigest enzymes with only a few recognition sites on the DNA are assayed using DNA cut with another restriction enzyme. FastDigest enzymes sensitive to Dam or Dcm methylation are assayed using DNA purified from dam, dcm strain of E.coli.
Ligation and Recleavage Assay

The ligation and recleavage assay confirms the integrity of DNA ends. DNA fragments obtained after overdigestion with 1 l of FastDigest enzyme for 1hour are ligated with T4 DNA ligase and then recut with the same enzyme. A FastDigest restriction enzyme conforms to the quality criterion if the observed ligation efficiency is identical to that of conventional enzyme. The percentage of DNA that can be successfully ligated and then re-cleaved for each FastDigest restriction enzyme is indicated in the in the product description and the Certificate of Analysis supplied with each enzyme.
Blue/White (B/W) Cloning Assay

The Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 1l of a FastDigest restriction enzyme in 1XFastDigest buffer. After a 1hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/Amp agar. An intact lacZ gene will give rise to a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxynucleases, the lacZ reading frame is interrupted, which results in the appearance of white colonies. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. For FastDigest restriction enzymes lacking recognition sites in pUC57 DNA, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3-overhang, 5-overhang and blunt ends).
Quality Control Labeled Oligonucleotide (LO) Test

1l of FastDigest enzyme is incubated with 5-[32P]-labeled single-stranded and double-stranded synthetic oligonucleotides in 1XFastDigest buffer for 1hour at the recommended temperature. The restriction enzyme passes this quality control test if there is no degradation of labeled oligonucleotides and no decrease in their specific radioactivity.
Prolonged Incubation / Star Activity Assay

The star activity assay is designed to test alterations in the DNA digestion pattern after prolonged incubation with FastDigest enzymes. 1 l of FastDigest enzyme is incubated with 1g of substrate DNA at the recommended temperature for up to 16hours. After electrophoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. The maximal incubation time that does not cause any star activity is indicated for each FastDigest enzyme in the product description and the Certificate of Analysis supplied with each enzyme.
Storage and Shipping

All FastDigest restriction enzymes should be stored at -20C. During shipment on dry ice, enzymes may freeze. This does not affect their quality all Fermentas enzymes are 100% active after at least three freeze-thaw cycles. For 24-48hour delivery, enzymes may be shipped on blue ice since their quality is not affected by short exposure to 4C.
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Product List
Table 1.2. FastDigest restriction enzymes.
RESTRICTION ENZYMES
FastDigest restriction enzyme FastDigest AatII FastDigest AccI (XmiI) FastDigest Acc65I FastDigest AciI (SsiI) FastDigest AclI (Psp1406I) FastDigest AcuI (Eco57I) FastDigest AfeI (Eco47III) FastDigest AflII (BspTI) FastDigest AgeI (BshTI) FastDigest AjuI FastDigest AleI (OliI) FastDigest AluI FastDigest Alw21I FastDigest Alw26I FastDigest AlwNI (CaiI) FastDigest ApaI FastDigest ApaLI (Alw44I) FastDigest AscI (SgsI) FastDigest AseI (VspI) FastDigest AsiSI (SfaAI) FastDigest AvaI (Eco88I) FastDigest AvaII (Eco47I) FastDigest AvrII (XmaJI) FastDigest BamHI FastDigest BanI (BshNI) FastDigest BbsI (BpiI) FastDigest BbvI (Lsp1109I) FastDigest BclI FastDigest BfaI (FspBI) FastDigest BglI FastDigest BglII FastDigest BlpI (Bpu1102I) FastDigest Bme1580I (BseSI) FastDigest BmtI (BspOI) FastDigest BplI FastDigest BpmI (GsuI) FastDigest Bpu10I FastDigest BsaAI (Ppu21I) FastDigest BsaBI (BseJI) FastDigest BsaHI (Hin1I) FastDigest BsaJI (BseDI) FastDigest BseGI FastDigest BseNI FastDigest BseXI FastDigest Bsh1236I FastDigest BsiEI (Bsh1285I) FastDigest BsiWI (Pfl23II) FastDigest BslI (BseLI)
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Specificity 53 GACGTC GTMKAC GGTACC CCGC(-3/-1) AACGTT CTGAAG(16/14) AGCGCT CTTAAG ACCGGT (7/12)GAA(N)7 TTGG(11/6) CACNNNNGTG AGCT GWGCWC GTCTC(1/5) CAGNNNCTG GGGCCC GTGCAC GGCGCGCC ATTAAT GCGATCGC CYCGRG GGWCC CCTAGG GGATCC GGYRCC GAAGAC(2/6) GCAGC(8/12) TGATCA CTAG GCCNNNNNGGC AGATCT GCTNAGC GKGCMC GCTAGC (8/13)GAG(N) 5CTC(13/8) CTGGAG(16/14) CCTNAGC(-5/-2) YACGTR GATNNNNATC GRCGYC CCNNGG GGATG(2/0) ACTGG(1/-1) GCAGC(8/12) CGCG CGRYCG CGTACG CCNNNNNNNGG
Prototype
Cat. # FD0994 FD1484 FD0904 FD1794 FD0944 FD0344 FD0324 FD0834 FD1464 FD1954 FD1634 FD0014 FD0024 FD0034 FD1394 FD1414 FD0044 FD1894 FD0914 FD2094 FD0384 FD0314 FD1564 FD0054/5 FD1004 FD1014 FD2074 FD0724 FD1764 FD0074 FD0083/4 FD0094 FD1444 FD2044 FD1314 FD0464 FD1184 FD1974 FD1414 FD0474 FD1084 FD0874 FD0884 FD1454 FD0924 FD0894 FD0854 FD1204
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AatII
AccI KpnI (GGTACC) AciI AclI Eco57I Eco47III AflII AgeI AjuI OliI AluI HgiAI BsmAI AlwNI ApaI ApaLI AscI VspI SgfI AvaI AvaII AvrII BamHI HgiCI BbvII BbvI BclI MaeI BglI BglII EspI BseSI NheI (GCTAGC) BplI BpmI Bpu10I BsaAI BsaBI AcyI SecI FokI (GGATG(9/13)) BsrI BbvI FnuDII McrI SplI BsiYI
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Table 1.2.FastDigest restriction enzymes.
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FastDigest restriction enzyme FastDigest BsmBI (Esp3I) FastDigest BsmFI (FaqI) FastDigest Bsp119I FastDigest Bsp120I FastDigest Bsp1286I (SduI) FastDigest Bsp1407I FastDigest BspCNI (BseMII) FastDigest BspHI (PagI) FastDigest BspMI (BveI) FastDigest BsrBI (MbiI) FastDigest BsrDI (BseMI) FastDigest BsrFI (Cfr10I) FastDigest BssHII (PteI) FastDigest BstXI FastDigest BstZ17I (Bst1107I) FastDigest Bsu36I (Eco81I) FastDigest ClaI (Bsu15I) FastDigest Csp6I FastDigest DdeI (HpyF3I) FastDigest DpnI FastDigest DraI FastDigest DraIII (AdeI) FastDigest DrdI (AasI) FastDigest EagI (Eco52I) FastDigest Eam1105I FastDigest EarI (Eam1104I) FastDigest Ecl136II FastDigest Eco31I FastDigest Eco91I FastDigest EcoNI (XagI) FastDigest EcoO109I FastDigest EcoRI FastDigest EcoRV (Eco32I) FastDigest EheI FastDigest Fnu4HI (SatI) FastDigest FokI FastDigest FspI (NsbI) FastDigest FspAI FastDigest HaeII (BfoI) FastDigest HaeIII (BsuRI) FastDigest HgaI (CseI) FastDigest HhaI FastDigest HincII FastDigest HindIII FastDigest HinfI FastDigest HinP1I (Hin6I) FastDigest HpaI (KspAI) FastDigest HpaII FastDigest Hpy8I FastDigest HpyF10VI
Prototype Esp3I FinI AsuII ApaI (GGGCCC) SduI Bsp1407I BseMII BspHI BspMI BsrBI BsrDI Cfr10I BsePI BstXI SnaI SauI ClaI RsaI (GTAC) DdeI DpnI AhaIII DraIII DrdI XmaIII Eam1105I Ksp632I SacI (GAGCTC) Eco31I BstEII EcoNI DraII EcoRI EcoRV NarI (GGCGCC) Fnu4HI FokI MstI FspAI HaeII HaeIII HgaI HhaI HindII HindIII HinfI HhaI (GCGC) HpaI HpaII MjaIV MwoI
Specificity 53 CGTCTC(1/5) GGGAC(10/14) TTCGAA GGGCCC GDGCHC TGTACA CTCAG(10/8) TCATGA ACCTGC(4/8) CCGCTC(-3/-3) GCAATG(2/0) RCCGGY GCGCGC CCANNNNNNTGG GTATAC CCTNAGG ATCGAT GTAC CTNAG Gm6ATC TTTAAA CACNNNGTG GACNNNNNNGTC CGGCCG GACNNNNNGTC CTCTTC(1/4) GAGCTC GGTCTC(1/5) GGTNACC CCTNNNNNAGG RGGNCCY GAATTC GATATC GGCGCC GCNGC GGATG(9/13) TGCGCA RTGCGCAY RGCGTY GGCC GACGC(5/10) GCGC GTYRAC AAGCTT GANTC GCGC GTTAAC CCGG GTNNAC GCNNNNNNNGC
Cat. # FD0454 FD1814 FD0124 FD0134 FD0654 FD0933/4 FD1404 FD1284 FD1744 FD1274 FD1264 FD0184 FD2134 FD1024 FD0704 FD0374 FD0143/4 FD0214 FD1884 FD1703/4 FD0224 FD1234 FD1724 FD0334 FD0244 FD0234 FD0254 FD0293/4 FD0394 FD1304 FD0264 FD0274/5 FD0303/4 FD0443/4 FD1644 FD2144 FD1224 FD1664 FD2184 FD0154 FD1904 FD1854 FD0494 FD0504/5 FD0804 FD0484 FD1034 FD0514 FD1574 FD1734
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RESTRICTION ENZYMES
FastDigest restriction enzyme FastDigest KpnI FastDigest Kpn2I FastDigest MauBI FastDigest MboI FastDigest MboII FastDigest MfeI (MunI) FastDigest MluI FastDigest MlyI (SchI) FastDigest MnlI FastDigest MreI FastDigest MscI (MlsI) FastDigest MseI (SaqAI) FastDigest MslI (RseI) FastDigest MspI FastDigest MssI FastDigest MvaI FastDigest Mva1269I FastDigest NaeI (PdiI) FastDigest NciI (BcnI) FastDigest NcoI FastDigest NdeI FastDigest NheI FastDigest NlaIII (Hin1II) FastDigest NlaIV (BspLI) FastDigest NmuCI FastDigest NotI FastDigest NruI (RruI) FastDigest NsiI (Mph1103I) FastDigest NspI (XceI) FastDigest PacI FastDigest PdmI FastDigest PflMI (Van91I) FastDigest PfoI FastDigest PmlI (Eco72I) FastDigest PpuMI (Psp5II) FastDigest PshAI (BoxI) FastDigest PsiI (AanI) FastDigest PspFI FastDigest PstI FastDigest PsuI FastDigest PsyI FastDigest PvuI FastDigest PvuII FastDigest RsaI FastDigest RsrII (CpoI) FastDigest SacI FastDigest SalI FastDigest SanDI (KflI) FastDigest SapI (LguI) FastDigest Sau3AI (Bsp143I)
Prototype KpnI BspMII MauBI MboI MboII MfeI MluI PleI (GAGTC(4/5) ) MnlI Sse232I BalI MseI MslI HpaII PmeI EcoRII (CCWGG) BsmI NaeI CauII NcoI NdeI NheI NlaIII NlaIV Tsp45I NotI NruI AvaIII NspI PacI XmnI PflMI PfoI PmaCI PpuMI PshAI PsiI BseYI PstI XhoII Tth111I PvuI PvuII RsaI RsrII SacI SalI SanDI SapI MboI
Specificity 53 GGTACC TCCGGA CGCGCGCG GATC GAAGA(8/7) CAATTG ACGCGT GAGTC(5/5) CCTC(7/6) CGCCGGCG TGGCCA TTAA CAYNNNNRTG CCGG GTTTAAAC CCWGG GAATGC(1/-1) GCCGGC CCSGG CCATGG CATATG GCTAGC CATG GGNNCC GTSAC GCGGCCGC TCGCGA ATGCAT RCATGY TTAATTAA GAANNNNTTC CCANNNNNTGG TCCNGGA CACGTG RGGWCCY GACNNNNGTC TTATAA CCCAGC(-5/-1) CTGCAG RGATCY GACNNNGTC CGATCG CAGCTG GTAC CGGWCCG GAGCTC GTCGAC GGGWCCC GCTCTTC(1/4) GATC
Cat. # FD0524 FD0534 FD2084 FD0814 FD0824 FD0753/4 FD0564 FD1374 FD1074 FD2024 FD1214 FD2174 FD2004 FD0544 FD1344 FD0554 FD0964 FD1524 FD0064 FD0573/4/5 FD0583/4/5 FD0973/4 FD1834 FD1154 FD1514 FD0593/4/6 FD2154 FD0734 FD1474 FD2204 FD1534 FD0714 FD1754 FD0364 FD0764 FD1434 FD2064 FD2224 FD0614/5 FD1554 FD1334 FD0624 FD0634 FD1124 FD0744 FD1133/4 FD0644 FD2164 FD1934 FD0784
Page 43 44 44 44 45 45 45 46 46 46 47 47 47 48 48 48 49 49 49 50 50 50 51 51 51 52 52 52 53 53 53 54 54 54 55 55 55 56 56 56 57 57 57 58 58 58 59 59 59 60
1
FastDigest restriction enzyme FastDigest Sau96I (Cfr13I) FastDigest SbfI (SdaI) FastDigest ScaI FastDigest ScrFI (Bme1390I) FastDigest SexAI (CsiI) FastDigest SfaNI (BmsI) FastDigest SfcI (BfmI) FastDigest SfiI FastDigest SmaI FastDigest SnaBI (Eco105I) FastDigest SpeI (BcuI) FastDigest SphI (PaeI) FastDigest SspI FastDigest StuI (Eco147I) FastDigest StyI (Eco130I) FastDigest SwaI (SmiI) FastDigest TaaI FastDigest TaiI FastDigest TaqI FastDigest TatI FastDigest TauI FastDigest TfiI (PfeI) FastDigest Tru1I FastDigest Tsp509I (TasI) FastDigest TspRI (TscAI) FastDigest XapI FastDigest XbaI FastDigest XhoI
Prototype AsuI Sse8387I ScaI ScrFI SexAI SfaNI SfeI SfiI SmaI SnaBI SpeI SphI SspI StuI StyI SwaI Tsp4CI MaeII (ACGT) TaqI TatI TauI TfiI MseI TspEI TspRI ApoI XbaI XhoI
Specificity 53 GGNCC CCTGCAGG AGTACT CCNGG ACCWGGT GCATC(5/9) CTRYAG GGCCNNNNNGGCC CCCGGG TACGTA ACTAGT GCATGC AATATT AGGCCT CCWWGG ATTTAAAT ACNGT ACGT TCGA WGTACW GCSGC GAWTC TTAA AATT CASTG(2/-7) RAATTY TCTAGA CTCGAG
Cat. # FD0194 FD1194 FD0434 FD1424 FD2114 FD2124 FD1164 FD1824 FD0663/4 FD0404 FD1253/4 FD0604 FD0774 FD0424 FD0414 FD1244 FD1364 FD1144 FD0674 FD1294 FD1654 FD1784 FD0984 FD1354 FD2104 FD1383/4 FD0684/5 FD0694/5
Page 60 60 61 61 61 62 62 62 63 63 63 64 64 64 65 65 65 66 66 66 67 67 67 68 68 68 69 69
Alphabetic list of commercially available restriction enzymes see p.207.
Single letter code

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10
1.
Product Description
RESTRICTION ENZYMES
FastDigest AatII
5...G pUC19 DNA/SmaI, 1.0% agarose 3...CT
1
Formulation Methylation Effects
Dam, Dcm, EcoKI, EcoBI no effect. CpG: completely overlaps blocked (p.178). 1l of enzyme (1 FDU) cleaves 1g of pUC19 DNA/SmaI fragments in 15min at 37C in 1XFastDigest Buffer.
A C G TC...3 G C A G...5 50l
#FD0994
Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Reaction Conditions
1 cleavage site
1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.
Lambda DNA, 1g/20l 15 min
Digestion time with 1l of FastDigest AatII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 20 min 15 min 15 min 5 bp 80C, 5 min 16hours
FastDigest AccI (XmiI)

5...G 3...C
TM K A C...3 A K MT G...5 #FD1484 50l

DNA, 1.0% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, Dcm, EcoKI, EcoBI no effect. CpG: may overlap blocked (p.178).
Reaction Conditions
9 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AccI (XmiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 60 min 5 min 2 bp 65C, 5 min 16hours
FastDigest Acc65I
3...C
T A C C...3 C A T GG...5 #FD0904 100l

5...GG
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/BamHI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, EcoKI, EcoBI no effect. Dcm, CpG: may overlap cleavage impaired (p.177, 179).
Reaction Conditions
2 cleavage sites
Note
FastDigest Acc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest Acc65I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
Protocols and Recommendations p.70
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1
FastDigest AciI (SsiI)

3...G
G C...3 G CG...5 #FD1794
5...CC
Formulation
20l
DNA, 2.0% agarose 516 cleavage sites
Methylation Effects
Dam, Dcm, EcoKI, EcoBI no effect. CpG: completely overlaps blocked (p.178).
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AciI (SsiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 4hours
FastDigest AclI (Psp1406I)

AC G T T...3 T G CA A...5 #FD0944 20l
5...A 3...T DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam, Dcm no effect. CpG: completely overlaps blocked (p.178). EcoKI, EcoBI: may overlap effect not determined.
Reaction Conditions
7 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AclI (Psp1406I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest AcuI (Eco57I)

T G A A G (N)16...3 A C T T C (N)14...5 #FD0344 20l
5...C 3...G DNA, 1.0% agarose 40 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 20XSAM (0.2 mM) 20l
Formulation
Note
FastDigest AcuI (Eco57I) requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates with FastDigestAcuI (Eco57I) is difficult to achieve. For cleavage with FastDigest AcuI (Eco57I) at least two copies of its recognition sequence are required. FastDigest AcuI (Eco57I) may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6XDNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Reaction Conditions
1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C. SAM 0.01 mM.
Methylation Effects
Dam, Dcm, CpG, EcoKI no effect. EcoBI: may overlap effect not determined.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AcuI (Eco57I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 30 min 15 min 15 min 2 bp 65C, 5 min 16hours
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RESTRICTION ENZYMES
FastDigest AfeI (Eco47III)

G CG C T...3 3...T C G C G A ...5 #FD0324 20l
5...A DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/Eco81I fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: completely overlaps blocked (p.178). EcoBI: may overlap effect not determined.
Reaction Conditions
2 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AfeI (Eco47III) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 5 min 16hours
FastDigest AflII (BspTI)

5...C T 3...G
T A A G...3 A A T T C...5 #FD0834 150l

Formulation
Methylation Effects
Dam, Dcm, CpG, EcoBI, EcoKI no effect.
Reaction Conditions
3 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AflII (BspTI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp No 16hours
FastDigest AgeI (BshTI)

3...T
C G G T...3 G G C CA...5 #FD1464 20l

DNA, 0.7% agarose 13 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
5...AC
Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AgeI (BshTI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 5 min 16hours
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1
FastDigest AjuI
3... 12(N)
G A A(N)7 T T G G (N)11...3 C T T(N)7 A A C C (N)6 ...5 #FD1954 20l

FX174 DNA, 1.0% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 20XSAM (0.2 mM) 20 l
5... 7(N)
Formulation
1l of enzyme (1 FDU) cleaves 1g of FX174 DNA in 5min at 37C in 1XFastDigest Buffer.
Note
FastDigest AjuI requires S-adenosylmethionine for activity. Still, complete cleavage of some substrates with FastDigest AjuI is difficult to achieve. FastDigest AjuI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.
Reaction Conditions
1 cleavage site
Methylation Effects
Dam, Dcm, CpG, EcoKI, EcoBI no effect.
Digestion time with 1l of FastDigest AjuI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 10 min 5 min ND 65C, 5 min 16hours
FastDigest AleI (OliI)

A C N NN N G T G...3 T G N NN N C A C...5 #FD1634 20l
5...C 3...G Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
FX174 DNA, 1.0% agarose
Methylation Effects
Dam, Dcm no effect. CpG: may overlap cleavage impaired (p.179). EcoKI, EcoBI: may overlap blocked (p.181).
Reaction Conditions
1 cleavage site
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AleI (OliI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 10 min 5 min 3 bp 65C, 5 min 6hours
FastDigest AluI
5...A 3...T
GC T...3 CG A...5 100l

Formulation
Methylation Effects
Dam, Dcm, CpG, EcoKI no effect. EcoBI: may overlap blocked (p.181).
#FD0014
Reaction Conditions
Digestion time with 1l of FastDigest AluI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 4 bp 65C, 5 min 16hours
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RESTRICTION ENZYMES
FastDigest Alw21I
5...G
W G C WC...3 3...C W C G W G ...5 100l

Formulation
Methylation Effects
#FD0024
Reaction Conditions
Digestion time with 1l of FastDigest Alw21I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 80C, 20 min 16hours
FastDigest Alw26I
3...C
T C T C(N)1...3 A G A G(N)5...5 #FD0034 100l

5...G DNA, 1.0% agarose 37 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam, Dcm, EcoKI, EcoBI no effect. CpG: may overlap cleavage impaired (p.179).
Reaction Conditions
Digestion time with 1l of FastDigest Alw26I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 16hours
FastDigest AlwNI (CaiI)

3...G
A G N N NC T G...3 T CN N N G A C...5 #FD1394 50l

5...C DNA, 0.7% agarose 41 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam, CpG, EcoKI no effect. Dcm: may overlap blocked (p.177). EcoBI: may overlap effect not determined.
Reaction Conditions
Note
FastDigest AlwNI (CaiI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AlwNI (CaiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 10 min 16hours
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1
FastDigest ApaI
5...G 3...C C
G G C CC...3 C G G G...5 300l

DNA, 0.7% agarose
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/CpoI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
#FD1414
Reaction Conditions
1 cleavage site
Note
FastDigest ApaI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest ApaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 20 min 10 min 2 bp 65C, 5 min 16hours
FastDigest ApaLI (Alw44I)

G C A C...3 A C G TG...5 #FD0044 200l
3...C DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...G T
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/SmaI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
4 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest ApaLI (Alw44I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 60 min 10 min 5 min 4 bp 80C, 5 min 16hours
FastDigest AscI (SgsI)

GC G C G C C...3 C G C G CG G...5 #FD1894 100l
5...G 3...C DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
2 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AscI (SgsI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 20 min 16hours
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RESTRICTION ENZYMES
FastDigest AseI (VspI)

TT A A T...3 3...T A A T T A ...5 #FD0914 200l
5...A DNA , 1.0% agarose 17 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AseI (VspI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 30 min 2 bp 65C, 5 min 16hours
FastDigest AsiSI (SfaAI)

pJET1-SfaAI DNA/SdaI, 1.0% agarose
C G A TC G C...3 G CT A G C G...5 #FD2094 100l

5...G 3...C Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of linearized pJET1 DNA with inserted SfaAI recognition site in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
1 cleavage site
Lambda DNA, 1g/20l no cleavage sites
Digestion time with 1l of FastDigest AsiSI (SfaAI) bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 60 min 5 min 5 bp 80C, 5 min 6hours
FastDigest AvaI (Eco88I)

3...G
C G R G...3 R G C YC...5 #FD0384 200l

5...CY
Formulation
Methylation Effects
Dam, Dcm, EcoKI, EcoBI no effect. CpG: completely overlaps cleavage impaired (p.178).
Reaction Conditions
8 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AvaI (Eco88I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 16hours
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1
FastDigest AvaII (Eco47I)

3...C
W C C ...3 C W GG...5 #FD0314
5...GG
Formulation
200l
Methylation Effects
Dam, EcoKI, EcoBI no effect. Dcm, CpG: may overlap blocked (p.177, 179).
Reaction Conditions
Note
FastDigest AvaII (Eco47I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AvaII (Eco47I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 80C, 20 min 16hours
FastDigest AvrII (XmaJI)

5...CC 3...G
T A G G...3 G A T CC...5 #FD1564 20l

Formulation
Methylation Effects
Reaction Conditions
2 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest AvrII (XmaJI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 5 min 10 min 2 bp No 16hours
FastDigest BamHI
5...G G 3...C
A T C C...3 C T A GG...5 800l

DNA, 0.7% agarose
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/Bsp120I fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
#FD0054
#FD0055
2500l
Supplied with: 10XFastDigest buffer 5x1 ml 10XFastDigest Green Buffer 5x1 ml
Digestion time with 1l of FastDigest BamHI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 1hour
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5 cleavage sites
Reaction Conditions
18
1.
RESTRICTION ENZYMES
FastDigest BanI (BshNI)

Y R C C...3 3...C C R Y G G ...5 #FD1004 300l
Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...G G
Formulation
DNA (dcm), 1.0% agarose
Methylation Effects
Dam, EcoBI no effect. Dcm, CpG: may overlap cleavage impaired (p.177, 179). EcoKI: may overlap effect not determined.
1l of enzyme (1 FDU) cleaves 1g of lambda DNA dcm in 5min at 37C in 1XFastDigest Buffer.
25 cleavage sites
Reaction Conditions
Note
FastDigest BanI (BshNI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BanI (BshNI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 60 min 5 min 15 min 2 bp 65C, 10 min 16hours
FastDigest BbsI (BpiI)

A A G A C(N)2...3 T T C T G(N)6...5 #FD1014 20l
5...G 3...C DNA. 0.7% agarose 24 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BbsI (BpiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 10 min 16hours
FastDigest BbvI (Lsp1109I)

5...G 3...C
C A G C (N)8 ...3 G T C G (N)12...5

pBR322 DNA, 0.7% agarose
Formulation
1l of enzyme (1 FDU) cleaves 1g of pBR322 DNA in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
#FD2074
100l
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BbvI (Lsp1109I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 2 bp 65C, 5 min 1hour
21 cleavage sites
Reaction Conditions
19
1
FastDigest BclI
A T C A...3 C T A GT...5 #FD0724 300l
3...A Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...TG
Formulation
DNA (dam ), 0.7% agarose
Methylation Effects
Dam: completely overlaps blocked (p.176). Dcm, CpG, EcoKI no effect. EcoBI: may overlap blocked (p.181).
1l of enzyme (1 FDU) cleaves 1g of lambda DNA dam in 5min at 37C in 1XFastDigest Buffer.
Reaction Conditions
8 cleavage sites
Note
FastDigest BclI is blocked by dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest BclI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 15 min 5 min 3 bp 80C, 20 min 16hours
FastDigest BfaI (FspBI)

3...G
A G...3 A TC...5 #FD1764
5...CT
Formulation
50l
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BfaI (FspBI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 5 min 20 min 3 bp 80C, 5 min 16hours
FastDigest BglI
5...G 3...C
C C N N N NN G G C...3 G G NN N N N C C G...5 200l

Formulation
Methylation Effects
#FD0074
Reaction Conditions
Digestion time with 1l of FastDigest BglI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 2hours
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20
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RESTRICTION ENZYMES
FastDigest BglII
5...A G
DNA, 0.7% agarose
A T C T...3 3...T C T A G A ...5 #FD0083 100l #FD0084 200l

Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam, Dcm, CpG, EcoKI no effect. EcoBI: may overlap cleavage impaired (p.181).
Reaction Conditions
6 cleavage sites
Digestion time with 1l of FastDigest BglII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 20 min 30 min 20 min 3 bp No 16hours
FastDigest BlpI (Bpu1102I)

3...C
CT N A G C...3 G A N TC G...5 #FD0094 50l

5...G DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
6 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BlpI (Bpu1102I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 16hours
FastDigest Bme1580I (BseSI)

5...G 3...CM
K G C MC...3 C G K G...5 50l

Formulation
Methylation Effects
Dam, Dcm, CpG, EcoBI no effect. EcoKI: may overlap cleavage impaired (p.181).
#FD1444
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Bme1580I (BseSI) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 30 min 3 bp 80C, 15 min 16hours
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1
FastDigest BmtI (BspOI)

5...G 3...CG
C T A GC...3 A T C G...5 20l

DNA, 0.7% agarose
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/HindIII fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
#FD2044
Reaction Conditions
1 cleavage site
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BmtI (BspOI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 15 min 5 min 3 bp 80C, 5 min 1hour
FastDigest BplI
5... 8(N) G 3... 13(N) C
A G(N)5 C T C(N)13...3 T C(N)5 G A G(N)8 ...5

DNA/XhoI, 0.7% agarose
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/XhoI fragments in 15min at 37C in 1XFastDigest Buffer.
Methylation Effects
#FD1314
20l
Digestion time with 1l of FastDigest BplI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 20 min 30 min 30 min ND 65C, 5 min 16hours
FastDigest BpmI (GsuI)

3...G
T G G A G (N)16...3 A C C T C (N)14...5 #FD0464 20l

1 cleavage site
Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 20XSAM (1.0 mM) 20 l
Note
FastDigest BplI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates with FastDigest BplI is difficult to achieve.
Reaction Conditions
Formulation
Methylation Effects
Dam, CpG, EcoKI, EcoBI no effect. Dcm: may overlap blocked (p.177).
Reaction Conditions
Note
FastDigest BpmI (GsuI) requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. FastDigest BpmI (GsuI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BpmI (GsuI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 30 min 15 min 15 min 2 bp 65C, 5 min 16hours
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22
1.
RESTRICTION ENZYMES
FastDigest Bpu10I
M13mp18 DNA, 0.7% agarose
CT N A G C...3 3...G G A N TC G ...5 #FD1184 20l

5...C Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of M13mp18 DNA in 15min at 37C in 1XFastDigest Buffer.
Methylation Effects
Note
For cleavage with FastDigest Bpu10I at least two copies of its recognition sequence are required.
Reaction Conditions
4 cleavage sites
Digestion time with 1l of FastDigest Bpu10I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 30 min 15 min 3 bp 80C, 5 min 1hour
FastDigest BsaAI (Ppu21I)

3...R
A CG T R...3 T GC A Y...5 #FD1974

5...Y
Formulation
50l
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsaAI (Ppu21I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 6hours
FastDigest BsaBI (BseJI)

5...G 3...C
21 cleavage sites
DNA ( dam ), 1.0% agarose
A T N NN N A T C...3 T A N NN N T A G...5 #FD1714 200l
Formulation
Methylation Effects
Dam: may overlap blocked (p.176). Dcm, EcoKI, CpG no effect. EcoBI: may overlap effect not determined.
Reaction Conditions
Note
FastDigest BsaBI (BseJI) is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsaBI (BseJI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 1hour
23
1
FastDigest BsaHI (Hin1I)

RC G Y C...3 Y G CR G...5 #FD0474 100 l
Formulation
Methylation Effects
Dam, EcoKI no effect. Dcm: may overlap cleavage impaired (p.177). CpG: completely overlaps blocked (p.178). EcoBI: may overlap effect not determined.
40 cleavage sites
Reaction Conditions
Note
FastDigest BsaHI (Hin1I) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsaHI (Hin1I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 16hours
FastDigest BsaJI (BseDI)

3...G
N N G G...3 G N N CC...5 #FD1084
5...CC
Formulation
20l
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsaJI (BseDI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 5 min 16hours
FastDigest BseGI
G A T G N N...3 C T A CN N ...5 #FD0874 100l
5...G 3...C DNA, 1.0% agarose 150 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest BseGI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 16hours
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24
1.
RESTRICTION ENZYMES
FastDigest BseNI
C T G G N...3 3...T G A C C N ...5
5...A
Formulation
100l
Methylation Effects
Dam, Dcm, CpG no effect. EcoKI, EcoBI: may overlap effect not determined.
#FD0884
Reaction Conditions
Digestion time with 1l of FastDigest BseNI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 16hours
FastDigest BseXI
3...C
Digestion time with 1l of FastDigest BseXI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 3 bp 80C, 15 min 16hours
FastDigest Bsh1236I
5...C 3...G
GC G...3 CG C...5 100l

21 cleavage sites
C A G C(N)8 ...3 G T C G(N)12...5 #FD1454 20l

5...G
Formulation
Methylation Effects
Reaction Conditions
Formulation
Methylation Effects
#FD0924
Reaction Conditions
Digestion time with 1l of FastDigest Bsh1236I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 80C, 10 min 16hours
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1
FastDigest BsiEI (Bsh1285I)

5...C 3...G
G R YC G...3 CY R G C...5 100l

Formulation
Methylation Effects
Dam: may overlap cleavage impaired (p.176). Dcm, EcoKI, EcoBI no effect. CpG: completely overlaps blocked (p.178).
#FD0894
Reaction Conditions
Note
FastDigest BsiEI (Bsh1285I) cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsiEI (Bsh1285I) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 15 min 3 bp 80C, 15 min 1hour
FastDigest BsiWI (Pfl23II)

T A C G...3 C A T GC...5 #FD0854
3...G 5...C G
Formulation
50l
DNA, 0.7% agarose
Methylation Effects
CpG: completely overlaps blocked (p.178). Dam, Dcm, EcoKI, EcoBI no effect.
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/Psp1406I fragments in 15min at 37C in 1XFastDigest Buffer.
Reaction Conditions
1 cleavage site
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsiWI (Pfl23II) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 15 min 3 bp 65C, 5 min 16hours
FastDigest BslI (BseLI)

5...C 3...G
C N N N N NN N G G...3 G N NN N N N N C C...5
Formulation
176 cleavage sites
Methylation Effects
#FD1204
100l
Reaction Conditions
Note
FastDigest BslI (BseLI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BslI (BseLI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp No 16hours
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26
1.
RESTRICTION ENZYMES
FastDigest BsmBI (Esp3I)

G T C T C(N)1...3 3...G C A G A G(N)5...5 #FD0454 20l
5...C DNA, 1.0% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
14 clavage sites
1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C. DTT (#R0861/2) 1 mM.
Note
The enzyme requires DTT (#R0861/2). Freshly made DTT should be added to the reaction buffer.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsmBI (Esp3I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 10 min 6hours
FastDigest BsmFI (FaqI)

3...C
G G A C(N)10...3 C C T G(N)14...5 #FD1814 20l

5...G DNA, 1.0% agarose 38 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 20XSAM (1.0 mM) 20 l
Formulation
Methylation Effects
Dam, Dcm, EcoBI, EcoKI no effect. CpG: may overlap blocked (p.179).
Reaction Conditions
Note
FastDigest BsmFI (FaqI) requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. Still, complete cleavage of some substrates is difficult to achieve. For cleavage with FastDigest BsmFI (FaqI) at least two copies of its recognition sequence are required.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsmFI (FaqI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 3 bp 65C, 5 min 16hours
FastDigest Bsp119I
3...A
TC G A A...3 A G CT T...5 #FD0124 200l

5...T DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam, Dcm, EcoBI no effect. CpG: completely overlaps blocked (p.178). EcoKI: may overlap effect not determined.
Reaction Conditions
7 cleavage sites
Digestion time with 1l of FastDigest Bsp119I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 80C, 5 min 16hours
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1
FastDigest Bsp120I
G C C C...3 C C G GG...5 #FD0134 200l
3...C DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...GG
Formulation
Methylation Effects
Reaction Conditions
1 cleavage site
Note
FastDigest Bsp120I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
FastDigest Bsp1286I (SduI)

5...G 3...CH
D G C HC...3 C G D G...5 50l

Formulation
Methylation Effects
#FD0654
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Bsp1286I (SduI) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 5 min 2 bp 80C, 15 min 1hour
FastDigest Bsp1407I
T A C A...3 C A T GT...5 #FD0933 50l #FD0934 100l
3...A Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...T G
Formulation
DNA, 0.7% agarose
Methylation Effects
Reaction Conditions
5 cleavage sites
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28
1.
RESTRICTION ENZYMES
FastDigest BspCNI (BseMII)

T C A G(N)10...3 3...G A G T C(N)8 ...5 #FD1404 20l
5...C DNA, 1.0% agarose 79 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 20XSAM (0.2 mM) 20 l
Formulation
Methylation Effects
Dam, Dcm, CpG, EcoBI, EcoKI no effect.
Note
FastDigest BspCNI (BseMII) requires S-adenosylmethionine for activity. Sinefungin can replace SAM in the restriction reaction. In this case DNA is not methylated and more than 95% of the ligated BspCNI (BseMII) fragments can be recut by this enzyme.
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BspCNI (BseMII) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 30 min 15 min 15 min 2 bp 80C, 5 min 16hours
FastDigest BspHI (PagI)

5...TC 3...A
A T G A...3 G T A CT...5 #FD1284
Formulation
50l
Methylation Effects
Dcm, CpG, EcoKI no effect. Dam, EcoBI: may overlap cleavage impaired (p.176, 181).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BspHI (PagI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 5 min 10 min 3 bp 80C, 5 min 0.5hour
FastDigest BspMI (BveI)

5...A 3...T
C C T G C(N)4...3 G G A C G(N)8...5 50l

3 cleavage sites
Reaction Conditions
Note
FastDigest BspHI (PagI) cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
Formulation
Methylation Effects
Dam, Dcm no effect. CpG: may overlap cleavage impaired (p.179). EcoBI, EcoKI: may overlap effect not determined.
#FD1744
Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 20XOligonucleotide (0.01 mM) 20 l
Reaction Conditions
1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C. Oligonucleotide 0.5M. sequence in the reaction mixture significantly improves cleavage of plasmid DNAs, especially of those with a single FastDigest BspMI (BveI) site. Still, complete cleavage of some substrates is difficult to achieve. FastDigest BspMI (BveI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6XDNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Note
At least two copies of FastDigest BspMI (BveI) recognition site are required for efficient cleavage. Inclusion of 0.5M oligonucleotide with the FastDigest BspMI (BveI) recognition
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BspMI (BveI) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 60 min 20 min 5 bp 65C, 5 min 16hours
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1
FastDigest BsrBI (MbiI)

3...G
C GC T C...3 G CG A G...5 #FD1274 100l

Formulation
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: completely overlaps cleavage impaired (p.178). EcoBI: may overlap effect not determined.
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsrBI (MbiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest BsrDI (BseMI)

3...C
C A A T G N N...3 G T T A CN N ...5 #FD1264 20l

Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsrDI (BseMI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 3 bp 80C, 5 min 16hours
FastDigest BsrFI (Cfr10I)

3...Y
C G G Y...3 G G C CR...5 #FD0184
5...RC
Formulation
50l
Methylation Effects
Reaction Conditions
Note
For cleavage with FastDigest BsrFI (Cfr10I) at least two copies of its recognition sequence are required.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BsrFI (Cfr10I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 10 min 5 min 2 bp No 2hours
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30
1.
RESTRICTION ENZYMES
FastDigest BssHII (PteI)

G C G C...3 3...C G C G CG...5 #FD2134
5...GC
Formulation
20l
DNA, 0.7% agarose
Methylation Effects
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BssHII (PteI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 5 min 16hours
FastDigest BstXI
C A N N N N NN T G G...3 G T NN N N N N A C C...5 #FD1024 100l
5...C 3...G DNA, 0.7% agarose 13 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
6 cleavage sites
Reaction Conditions
Formulation
Methylation Effects
Dam, CpG, EcoKI, EcoBI no effect. Dcm: may overlap cleavage impaired (p.177).
Reaction Conditions
Note
FastDigest BstXI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest BstXI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 80C, 5 min 0.5hours
FastDigest BstZ17I (Bst1107I)

5...G 3...C
T AT A C...3 A TA T G...5 #FD0704 100l

Formulation
Methylation Effects
Reaction Conditions
3 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest BstZ17I (Bst1107I) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 10 min 3 bp No 6hours
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1
FastDigest Bsu36I (Eco81I)

3...G
CT N A G G...3 G A N TC C...5 #FD0374 50l

5...C DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
2 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Bsu36I (Eco81I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 10 min 16hours
FastDigest ClaI (Bsu15I)

3...T
DNA, 0.7% agarose
TC G A T...3 A G CT A...5 #FD0143 50l #FD0144 100l

5...A Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam: may overlap blocked (p.176). CpG: completely overlaps blocked (p.178). Dcm, EcoKI no effect. EcoBI: may overlap effect not determined.
Reaction Conditions
15 cleavage sites
Note
FastDigest ClaI (Bsu15I) is blocked by overlapping dam methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest ClaI (Bsu15I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 15 min 16hours
FastDigest Csp6I
A C...3 A TG...5 #FD0214
3...C 5...GT
Formulation
100l
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest Csp6I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 10 min 16hours
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32
1.
RESTRICTION ENZYMES
FastDigest DdeI (HpyF3I)

N A G...3 3...G A N T C...5 #FD1884
5...C T
Formulation
50l
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest DdeI (HpyF3I) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 10 min 3 bp 65C, 5 min 16hours
FastDigest DpnI
5...G 3...C
m6A T C...3 Tm6A G...5

Formulation
50l 100l 1l of enzyme (1 FDU) cleaves 1g of pBR322 DNA (dam methylated) in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam: does not cut dam DNA (p.176). Dcm, EcoKI, CpG no effect. EcoBI: may overlap effect not determined.
#FD1703 #FD1704
22 cleavage sites
Reaction Conditions
1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C. FastDigest DpnI will only cleave fullyadenomethylated dam sites. FastDigest DpnI, FastDigest Sau3AI (Bsp143I) and FastDigest MboI all recognize the same sequence but have different methylation sensitivities (pp.176181) and cleavage positions.
Note
FastDigest DpnI requires the presence of N6-methyladenine within the recognition sequence to cleave DNA. DNA purified from a dam+ strain will be a substrate for FastDigest DpnI.
Lambda DNA, 1g/20l not determined
Digestion time with 1l of FastDigest DpnI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min not applicable 10 min 1 bp 80C, 5 min 16hours
FastDigest DraI
T TA A A...3 A AT T T...5 #FD0224 200l
5...T 3...A DNA, 0.7% agarose 13 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Dam, Dcm, CpG, EcoBI no effect. EcoKI: may overlap blocked (p.181).
Reaction Conditions
Digestion time with 1l of FastDigest DraI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 5 min 16hours
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1
FastDigest DraIII (AdeI)

3...G
A C N N NG T G...3 T GN N N C A C...5 #FD1234 50l

Formulation
Methylation Effects
Dam, Dcm no effect. CpG: may overlap cleavage impaired (p.179). EcoKI, EcoBI: may overlap effect not determined.
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest DraIII (AdeI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 6hours
FastDigest DrdI (AasI)

3...C
A C N N N NN N G T C...3 T G N NN N N N C A G...5 #FD1724 25l

Formulation
Methylation Effects
Dam, Dcm, EcoBI, EcoKI no effect. CpG: may overlap cleavage impaired (p.179).
Reaction Conditions
3 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest DrdI (AasI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 80C, 10 min 1hour
FastDigest EagI (Eco52I)

3...G
G C C G...3 C C G GC...5 #FD0334
5...CG
Formulation
50l
DNA, 0.7% agarose
Methylation Effects
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest EagI (Eco52I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 20 min 20 min 5 min 3 bp 65C, 5 min 16hours
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2 cleavage sites
Reaction Conditions
34
1.
RESTRICTION ENZYMES
FastDigest Eam1105I
A C N N NN N G T C...3 3...C T G N N N N N C A G ...5 #FD0244 100l
Formulation
Methylation Effects
Reaction Conditions
9 cleavage sites
Digestion time with 1l of FastDigest Eam1105I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest EarI (Eam1104I)

T C T T C(N)1...3 A G A A G(N)4...5 #FD0234 50l
Formulation
Methylation Effects
Reaction Conditions
2 cleavage sites
Note
Certain sites in DNA are difficult to cleave with FastDigest EarI (Eam1104I), the same as with its prototype Ksp632I.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest EarI (Eam1104I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 5 min 6hours
FastDigest Ecl136II
A GC T C...3 T CG A G...5 #FD0254 100l
Formulation
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: may overlap cleavage impaired (p.179). EcoBI: may overlap effect not determined.
Reaction Conditions
2 cleavage sites
Digestion time with 1l of FastDigest Ecl136II bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 6hours
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1
FastDigest Eco31I
G T C T C(N)1...3 C A G A G(N)5...5 #FD0293 50l #FD0294 100l
5...G 3...C Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
DNA (dcm ), 0.7% agarose
Methylation Effects
Dam, EcoKI no effect. Dcm, CpG: may overlap cleavage impaired (p.177, 179). EcoBI: may overlap effect not determined.
1l of enzyme (1 FDU) cleaves 1g of lambda DNA dcm/HindIII fragments in 5min at 37C in 1XFastDigest Buffer.
Reaction Conditions
2 cleavage sites
Note
FastDigest Eco31I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest Eco31I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest Eco91I
3...C
T N A C C...3 C A N T GG...5 #FD0394 200l

5...GG
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest Eco91I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 10 min 2hours
FastDigest EcoNI (XagI)

3...G
C T N NN N N A G G...3 G A N N NN N T C C...5 #FD1304 100l

5...C DNA, 0.7% agarose Supplied with: 1 ml 10XFastDigest Buffer 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
9 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest EcoNI (XagI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 20 min 2 bp 65C, 5 min 16hours
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36
1.
RESTRICTION ENZYMES
FastDigest EcoO109I
GG N C C Y...3 3...Y C C N GG R...5 #FD0264 200l
5...R DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
3 clevage sites
Note
FastDigest EcoO109I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest EcoO109I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 15 min 5 min 2 bp 65C, 5 min 16hours
FastDigest EcoRI
3...C
A T T C...3 T T A AG...5 #FD0274 800l

DNA, 0.7% agarose Supplied with: 10XFastDigest buffer 2x1 ml 10XFastDigest Green Buffer 2x1 ml Supplied with: 10XFastDigest buffer 5x1 ml 10XFastDigest Green Buffer 5x1 ml
5...GA
Formulation
Methylation Effects
Reaction Conditions
5 cleavage sites
#FD0275
2500l
Digestion time with 1l of FastDigest EcoRI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 20 min 5 min 2 bp 80C, 5 min 0.5hour
FastDigest EcoRV (Eco32I)

5...G 3...C
DNA, 1.0% agarose
A TA T C...3 T AT A G...5 #FD0303 200l #FD0304 400l

Formulation
Methylation Effects
Reaction Conditions
21 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest EcoRV (Eco32I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp No 16hours
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FastDigest EheI
G CG C C...3 C GC G G...5 #FD0443 20l #FD0444 50l
5...G 3...C Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/PstI fragments in 5min at 37C in 1XFastDigest Buffer.
DNA, 0.7% agarose
Methylation Effects
Reaction Conditions
1 cleavage site
Digestion time with 1l of FastDigest EheI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 6hours
FastDigest Fnu4HI (SatI)

5...G 3...C
CN G C...3 G NC G...5 20l

Formulation
Methylation Effects
#FD1644
Reaction Conditions
Note
At least two copies of FastDigest Fnu4HI (SatI) recognition site are required for an efficient cleavage.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Fnu4HI (SatI) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 5 min 3 bp 65C, 5 min 16hours
FastDigest FokI
5...G 3...C
G A T G(N)9...3 C T A C(N)13...5 #FD2144 100l

DNA, 1.0% agarose 150 cleavage sites Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Note
At least two copies of FastDigest FokI recognition site are required for an efficient cleavage. FastDigest FokI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Reaction Conditions
Methylation Effects
Digestion time with 1l of FastDigest FokI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 1hour
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38
1.
RESTRICTION ENZYMES
FastDigest FspI (NsbI)

G CG C A...3 3...A C G C G T ...5 #FD1224 50l
5...T DNA, 0.7% agarose 15 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest FspI (NsbI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 15 min 16hours
FastDigest FspAI
3...Y
T G CG C A Y...3 A C GC G T R...5 #FD1664 20l

5...R DNA, 0.5% agarose 2 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA/Psp1406I fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest FspAI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 5 min 16hours
FastDigest HaeII (BfoI)

5...R 3...YC
G C G CY...3 G C G R...5 200l

Formulation
1l of enzyme (1 FDU) cleaves 1 g of lambda DNA in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: completely overlaps blocked (p.178). EcoBI: may overtlap not determined.
#FD2184
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest HaeII (BfoI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 10 min 1hour
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FastDigest HaeIII (BsuRI)

5...G 3...C
GC C...3 CG G...5 400l

Formulation
Methylation Effects
#FD0154
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest HaeIII (BsuRI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 16hours
FastDigest HgaI (CseI)

3...C
11 cleavage sites
A C G C(N)5 ...3 T G C G(N)10...5 #FD1904 20l

5...G
Formulation
Note
For cleavage with FastDigest HgaI (CseI) at least two copies of its recognition sequence are required. FastDigest HgaI (CseI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Reaction Conditions
Methylation Effects
Dam, Dcm, EcoKI: never overlaps no effect. CpG: completely overlaps blocked (p.178). EcoBI: may overlap effect not determined.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest HgaI (CseI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 30 min 15 min 2 bp 80C, 5 min 16hours
FastDigest HhaI
5...G 3...C G
C GC...3 C G...5 200l

Formulation
Methylation Effects
#FD1854
Reaction Conditions
Digestion time with 1l of FastDigest HhaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp No 16hours
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40
1.
RESTRICTION ENZYMES
FastDigest HincII
T YR A C...3 3...C A R Y T G...5 #FD0494 100l
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest HincII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 1 bp 65C, 5 min 16hours
FastDigest HindIII
3...T
G C T T...3 T C G AA...5 #FD0504 800l

DNA, 0.7% agarose Supplied with: 10XFastDigest buffer 2x1 ml 10XFastDigest Green Buffer 2x1 ml Supplied with: 10XFastDigest buffer 5x1 ml 10XFastDigest Green Buffer 5x1 ml
5...AA
Formulation
Methylation Effects
Reaction Conditions
7 cleavage sites
#FD0505
2500l
Digestion time with 1l of FastDigest HindIII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 20 min 10 min 3 bp 80C, 10 min 16hours
FastDigest HinfI
N T C...3 T N AG...5 #FD0804
3...C 5...G A
Formulation
400l
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: may overlap cleavage impaired (p.179). EcoBI: may overlap blocked (p.181).
Reaction Conditions
Digestion time with 1l of FastDigest HinfI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 20 min 16hours
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FastDigest HinP1I (Hin6I)

3...C
G C...3 G CG...5 #FD0484
5...G C
Formulation
200l
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest HinP1I (Hin6I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 5 min 5 min 2 bp 80C, 10 min 16hours
FastDigest HpaI (KspAI)

T TA A C...3 A AT T G...5 #FD1034 50l
Formulation
Methylation Effects
Dam, Dcm, EcoBI no effect. CpG: may overlap cleavage impaired (p.179). EcoKI: may overlap blocked (p.181).
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest HpaI (KspAI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 20 min 0.5hour
FastDigest HpaII
3...G
G G...3 G CC...5 #FD0514
5...CC
Formulation
200l
Methylation Effects
Supplied with: 1 ml 10XFastDigest Buffer 10XFastDigest Green Buffer 1 ml
Reaction Conditions
Digestion time with 1l of FastDigest HpaII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
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1.
RESTRICTION ENZYMES
FastDigest Hpy8I
T NN A C...3 3...C A N N T G ...5 #FD1574 20l
Formulation
Methylation Effects
Dam, Dcm, no effect. CpG: may overlap cleavage impaired (p.179). EcoKI, EcoBI effect not determined.
Reaction Conditions
Digestion time with 1l of FastDigest Hpy8I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 2 bp 80C, 5 min 16hours
FastDigest HpyF10VI
3...C
C N N N N NN N G C...3 G N NN N N N N C G...5 #FD1734 20l

Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest HpyF10VI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 16hours
FastDigest KpnI
5...G 3...CC
G T A CC...3 A T G G...5 300l

DNA, 0.7% agarose
Formulation
Methylation Effects
Dam, Dcm, CpG, EcoKI, EcoKI, EcoBI no effect.
#FD0524
Digestion time with 1l of FastDigest KpnI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 5 min 16hours
2 cleavage sites
Reaction Conditions
43
1
FastDigest Kpn2I
3...A
C G G A...3 G G C CT...5 #FD0534 100l

5...TC
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest Kpn2I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 16hours
FastDigest MauBI
pJET1-MauBI DNA/BamHI, 0.7% agarose
GC G C G C G...3 C G C G CG C...5 #FD2084 20l

Formulation
1l of enzyme (1 FDU) cleaves 1g of linearized pJET1 DNA with inserted MauBI recognition sites in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
2 cleavage sites
Digestion time with 1l of FastDigest MauBI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 10 min 5 min 5 bp 65C, 5 min 16hours
FastDigest MboI
3...
116 cleavage sites
A T C ...3 C T A G...5 #FD0814
5...G
Formulation
50l 1l of enzyme (1 FDU) cleaves 1g of lambda DNA dam in 5min at 37C in 1XFastDigest Buffer.
Note
FastDigest MboI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099). FastDigest MboI, FastDigest Sau3AI (Bsp143I) and FastDigest DpnI all recognize the same sequence but have different methylation sensitivities (pp.176181) and cleavage positions.
Reaction Conditions
Methylation Effects
Dam: completely overlaps blocked (p.176). EcoBI: may overlap blocked (p.181). Dcm, CpG, EcoKI no effect.
Digestion time with 1l of FastDigest MboI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 1 bp 65C, 15 min 16hours
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44
1.
RESTRICTION ENZYMES
FastDigest MboII
A A G A(N)8...3 3...C T T C T(N)7...5 #FD0824 50l
5...G Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
130 cleavage sites
Note
FastDigest MboII is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099). FastDigest MboII produces DNA fragments that have a single-base 3extension which are more difficult to ligate than blunt-ended fragments. FastDigest MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
Reaction Conditions
Methylation Effects
Dam: may overlap blocked (p.176). Dcm, CpG, EcoKI, EcoBI no effect.
Digestion time with 1l of FastDigest MboII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 16hours
FastDigest MfeI (MunI)

3...G
DNA, 0.7% agarose
A T T G...3 T T A AC...5 #FD0753 #FD0754
5...CA
Formulation
20l 50l 1l of enzyme (1 FDU) cleaves 1g of lambda DNA in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
8 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest MfeI (MunI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 16hours
FastDigest MluI
3...T
G C G T...3 G C G CA...5 #FD0564 100l

5...AC
Formulation
Methylation Effects
Reaction Conditions
7 cleavage sites
Digestion time with 1l of FastDigest MluI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 15 min 3 bp 80C, 5 min 16hours
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FastDigest MlyI (SchI)

5...G
A G T C(N)5...3 T C A G(N)5...5 #FD1374 100l

Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest MlyI (SchI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 30 min 5 min 2 bp 80C, 5 min 1hour
FastDigest MnlI
C T C(N)7...3 G A G(N)6...5 #FD1074 50l
Formulation
Methylation Effects
Reaction Conditions
Note
FastDigest MnlI produces DNA fragments that have a single-base 3extension which are more difficult to ligate than blunt-ended fragments.
Digestion time with 1l of FastDigest MnlI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 16hours
FastDigest MreI
pJET1-MreI DNA/Eam1105I, 1.0% agarose 3...G
G C C G G C G...3 C G G C CG C...5 #FD2024 20l

5...C Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of linearized pJET1 DNA with inserted MreI recognition site in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
1 cleavage site
Digestion time with 1l of FastDigest MreI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 5 min 16hours
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46
1.
RESTRICTION ENZYMES
FastDigest MscI (MlsI)

DNA (dcm ), 0.7% agarose
G GC C A...3 3...A C C G G T ...5 #FD1214 50l

5...T Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
18 cleavage sites
Note
FastDigest MscI (MlsI) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest MscI (MlsI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 30 min 20 min 3 bp 80C, 20 min 16hours
FastDigest MseI (SaqAI)

5...T T 3...A
A A...3 A TT...5 #FD2174
Formulation
50l 1l of enzyme (1 FDU) cleaves 1g of pBR322 DNA in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, Dcm, CpG, EcoBI no effect. EcoKI: may overlap effect not determined.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest MseI (SaqAI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 5 min 16hours
FastDigest MslI (RseI)

3...G
A Y N N N N R T G...3 T R N NN N Y A C...5 #FD2004 20l

15 cleavage sites
Reaction Conditions
Formulation
Methylation Effects
Dam, Dcm, CpG no effect. EcoKI: may overlap blocked (p.181). EcoBI: may overlap effect not determined.
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest MslI (RseI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 20 min 16hours
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FastDigest MspI
3...G
G G...3 G CC...5 #FD0544
5...CC
Formulation
400l
Methylation Effects
Reaction Conditions
Note
FastDigest MspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methylated, these enzymes cannot cleave. However, unlike HpaII, FastDigest MspI can cleave the sequence when the internal C residue is methylated.
Digestion time with 1l of FastDigest MspI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 16hours
FastDigest MssI
T T TA A A C...3 A A AT T T G...5 #FD1344 100l
Formulation
Methylation Effects
Reaction Conditions
2 cleavage sites
Digestion time with 1l of FastDigest MssI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 10 min 16hours
FastDigest MvaI
5...C 3...G
CW G G...3 G WC C...5 200l

Formulation
Methylation Effects
#FD0554
Reaction Conditions
Digestion time with 1l of FastDigest MvaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 4 bp No 1hour
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1.
RESTRICTION ENZYMES
FastDigest Mva1269I
A A T G C N...3 3...C T T A CG N ...5 #FD0964 50l
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest Mva1269I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest NaeI (PdiI)

pBR322 DNA/NdeI, 0.7% agarose 3...C
C CG G C...3 G GC C G...5 #FD1524 50l

5...G Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of pBR322 DNA/NdeI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
4 cleavage sites
Note
For cleavage with FastDigest NaeI (PdiI) at least two copies of its recognition sequence are required. Certain sites in pBR322 are difficult to cleave with FastDigest NaeI (PdiI), the same as with its prototype NaeI.
Lambda DNA, 1g/20l not determined
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NaeI (PdiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 15 min 16hours
FastDigest NciI (BcnI)

5...C 3...G
CS G G...3 G SC C...5 200l

Formulation
Methylation Effects
#FD0064
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NciI (BcnI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 20 min 16hours
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1
FastDigest NcoI
3...G
DNA, 0.7% agarose
A T G G...3 G T A CC...5 #FD0573 20l #FD0574 100l #FD0575 300l

All supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
5...CC
Formulation
Methylation Effects
Reaction Conditions
4 cleavage sites
Digestion time with 1l of FastDigest NcoI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 10 min 5 min 3 bp 65C, 15 min 16hours
FastDigest NdeI
AT A T G...3 T A TA C...5 #FD0583 100l #FD0584 300l
5...C 3...G Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
DNA, 0.7% agarose
Methylation Effects
Reaction Conditions
7 cleavage sites
#FD0585
1000l
Supplied with: 10XFastDigest Buffer 2X1 ml 10XFastDigest Green Buffer 2X1 ml
Digestion time with 1l of FastDigest NdeI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 60 min 30 min 3 bp 65C, 5 min 6hours
FastDigest NheI
3...C
Digestion time with 1l of FastDigest NheI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 5 min 5 min 5 bp 65C, 5 min 6hours
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1 cleavage site
DNA 0.7% agarose
T A G C...3 G A T CG...5 #FD0973 50l #FD0974 100l
5...GC
Formulation
Methylation Effects
Reaction Conditions
50
1.
RESTRICTION ENZYMES
FastDigest NlaIII (Hin1II)

C A T G ...3 3... G T A C ...5
5...
Formulation
100l
Methylation Effects
#FD1834
Reaction Conditions
Note
More stable when stored at -70C. At -20C the half-life of FastDigest NlaIII (Hin1II) is 6 months.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NlaIII (Hin1II) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 5 min 10 min 4 bp 80C, 5 min 16hours
FastDigest NlaIV (BspLI)

G NN C C...3 C NN G G...5 #FD1154 20l
Formulation
Methylation Effects
Reaction Conditions
Note
FastDigest NlaIV (BspLI) cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NlaIV (BspLI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 20 min 16hours
FastDigest NmuCI
T S A C ...3 C A S T G...5 #FD1514
3... 5...G
Formulation
20l
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest NmuCI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 2 bp 65C, 5 min 16hours
51
1
FastDigest NotI
pTZ19RJL2/BseLI DNA, 1.0% agarose 3...C
CG G C C G C...3 G C C G GC G...5 #FD0593 20l #FD0594 50l #FD0596 250l

5...G All supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of pTZ19RJL2 DNA/BseLI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
1 cleavage site
Digestion time with 1l of FastDigest NotI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 30 min 5 min 10 min 2 bp 80C, 5 min 16hours
FastDigest NruI (RruI)

3...A
C G C G A...3 G C G C T...5 #FD2154 100l

Formulation
Methylation Effects
Dam, Dcm, EcoBI, EcoKI no effect. CpG: completely overlaps blocked (p.178).
Reaction Conditions
5 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NruI (RruI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 5 bp No 4hours
FastDigest NsiI (Mph1103I)

5...A 3...TA
T G C AT...3 C G T A...5 100l

Formulation
Methylation Effects
#FD0734
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NsiI (Mph1103I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 3 bp 65C, 15 min 6hours
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52
1.
RESTRICTION ENZYMES
FastDigest NspI (XceI)

C A T GY...3 3...YG T A C R...5
5...R
Formulation
50l
Methylation Effects
Dam, Dcm, EcoKI, EcoBI, CpG no effect.
#FD1474
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest NspI (XceI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 16hours
FastDigest PacI
pJET1-PacI DNA/SdaI, 1.0% agarose 3...A
T A A TT A A...3 A TT A A T T...5 #FD2204 25l

5...T Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of linearized pJET1 DNA with inserted PacI recognition site in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, Dcm, EcoBI, CpG no effect. EcoKI: may overlap effect not determined.
Reaction Conditions
1 cleavage site
Digestion time with 1l of FastDigest PacI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 10 min 16hours
FastDigest PdmI
A A N NN N T T C...3 T T N NN N A A G...5 #FD1534 100l
5...G 3...C DNA, 0.7% agarose 24 cleavage sites Supplied with: 1 ml 10XFastDigest Buffer 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest PdmI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 16hours
53
1
FastDigest PflMI (Van91I)

3...G
C A N N N NN T G G...3 G T NN N N N A C C...5 #FD0714 100l

Formulation
Methylation Effects
Reaction Conditions
Note
FastDigest PflMI (Van91I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest PflMI (Van91I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 10 min 6hours
FastDigest PfoI
3...A
C N G G A...3 G G N C CT...5 #FD1754 20l

5...TC
Formulation
Methylation Effects
Dam: may overlap cleavage impaired (p.176). Dcm, CpG: may overlap blocked (p.177, 179). EcoKI, EcoBI no effect.
Reaction Conditions
Note
FastDigest PfoI cleavage is impaired by overlapping dam methylation and blocked by overlapping dcm methylation. To avoid dam or dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest PfoI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest PmlI (Eco72I)

A CG T G...3 T GC A C...5 #FD0364 200l
5...C 3...G DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
3 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest PmlI (Eco72I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 10 min 1hour
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54
1.
RESTRICTION ENZYMES
FastDigest PpuMI (Psp5II)

GG W C C Y...3 3...Y C C W GG R...5 #FD0764 50l
5...R DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
3 cleavage sites
Note
FastDigest PpuMI (Psp5II) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest PpuMI (Psp5II) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 20 min 2 bp 80C, 5 min 1hour
FastDigest PshAI (BoxI)

A C N NN N G T C...3 T G N NN N C A G...5 #FD1434 100l
Formulation
Methylation Effects
Reaction Conditions
7 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest PshAI (BoxI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 20 min 16hours
FastDigest PsiI (AanI)

3...A
T AT A A ...3 A TA T T ...5 #FD2064 20l

Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest PsiI (AanI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 5 bp 65C, 5 min 16hours
55
1
FastDigest PspFI
5...C 3...G G
C C A GC...3 G T C G...5 20l

Formulation
Methylation Effects
#FD2224
Reaction Conditions
Digestion time with 1l of FastDigest PspFI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 80C, 5 min 16hours
FastDigest PstI
5...C 3...G A
T G C AG...3 C G T C...5 800l

Formulation
Note
Surrounding sequences: the presence of adjacent runs of G-C base pairs confers significant resistance to cleavage (Armstrong, K. and Bauer, W.R., NAR, 10, 993-1007, 1982). FastDigest PstI will not cut AGCTGCAG when methylated by AluI methyltransferase.
#FD0614
Reaction Conditions
#FD0615
2500l
Methylation Effects
Digestion time with 1l of FastDigest PstI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 30 min 5 min 3 bp No 16hours
FastDigest PsuI
5...R G 3...Y
A T C Y...3 C T A GR...5 150l

Formulation
Methylation Effects
#FD1554
Reaction Conditions
Digestion time with 1l of FastDigest PsuI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 16hours
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56
1.
RESTRICTION ENZYMES
FastDigest PsyI
A C NN N G T C...3 3...C T G N N N C A G ...5
5...G
Formulation
DNA, 0.7% agarose
Methylation Effects
#FD1334
100l
Digestion time with 1l of FastDigest PsyI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 80C, 5 min 4hours
FastDigest PvuI
5...C 3...G
G A TC G...3 CT A G C...5 20l

DNA, 0.7% agarose
2 cleavage sites
Reaction Conditions
Formulation
Methylation Effects
#FD0624
Digestion time with 1l of FastDigest PvuI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 5 min 5 min 4 bp 80C, 5 min 16hours
FastDigest PvuII
A GC T G...3 T CG A C...5 #FD0634 400l
3 cleavage sites
Reaction Conditions
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest PvuII bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp No 0.5hour
57
1
FastDigest RsaI
5...G 3...C
TA C...3 AT G...5 100l

Formulation
Methylation Effects
#FD1124
Reaction Conditions
Digestion time with 1l of FastDigest RsaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 16hours
FastDigest RsrII (CpoI)

5...C 3...G
G G W C C G...3 C C W G G C...5 20l

DNA, 0.7% agarose
Formulation
Methylation Effects
#FD0744
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest RsrII (CpoI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 16hours
FastDigest SacI
5...G 3...C T
A G C TC...3 C G A G...5 100l 200l
5 cleavage sites
Reaction Conditions
Formulation
DNA, 0.7% agarose
Methylation Effects
#FD1133 #FD1134
2 cleavage sites
Reaction Conditions
Note
FastDigest SacI is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block FastDigest SacI.
FastDigest SacI is inhibited by common clinical anticoagulants found in some preparations of anticoagulated peripheral blood and bone marrow. (Coad, J.E., et al., Inhibition of restriction endonucleases by common clinical anticoagulants, Anal. Biochem., 205, 368-369,1992).
Digestion time with 1l of FastDigest SacI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 30 min 10 min 1 bp 65C, 5 min 16hours
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58
1.
RESTRICTION ENZYMES
FastDigest SalI
C G A C...3 3...C A G C TG ...5 #FD0644 200l
DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...GT
Formulation
Methylation Effects
Reaction Conditions
2 cleavage sites
Digestion time with 1l of FastDigest SalI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 60 min 5 min 3 bp 65C, 10 min 16hours
FastDigest SanDI (KflI)

pJET1-SanDI DNA/BamHI, 0.7% agarose
G G W C C C...3 C C W G G G...5 #FD2164 20l

Formulation
1l of enzyme (1 FDU) cleaves 1g of linearized pJET1 DNA with inserted SanDI recognition sites in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Dam, EcoKI, EcoBI no effect. Dcm: may overlap effect not determined. CpG: may overlap cleavage impaired (p.179).
Reaction Conditions
3 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SanDI (KflI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 5 bp No 16hours
FastDigest SapI (LguI)

3...C
C T C T T C(N)1...3 G A G A A G(N)4...5 #FD1934 20l

Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SapI (LguI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 5 min 16hours
59
1
FastDigest Sau3AI (Bsp143I)

3...
A T C ...3 C T A G...5 #FD0784
5...G
Formulation
40l
Methylation Effects
Reaction Conditions
Note
FastDigest DpnI, FastDigest Sau3AI (Bsp143I) and FastDigest MboI all recognize the same sequence but have different methylation sensitivities (pp.176181) and cleavage positions.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Sau3AI (Bsp143I) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 5 min 4 bp 65C, 20 min 16hours
FastDigest Sau96I (Cfr13I)

5...GG 3...C
N C C...3 C N GG...5 #FD0194
Formulation
100l
Methylation Effects
Reaction Conditions
Note
FastDigest Sau96I (Cfr13I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Sau96I (Cfr13I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp No 16hours
FastDigest SbfI (SdaI)

5...C 3...G
C T G C AG G...3 GA C G T C C...5 20l

DNA, 0.7% agarose
Formulation
Methylation Effects
#FD1194
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SbfI (SdaI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 1hour
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5 cleavage sites
Reaction Conditions
60
1.
RESTRICTION ENZYMES
FastDigest ScaI
G TA C T...3 3...T C A T G A ...5 #FD0434 100l
5...A DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
Methylation Effects
Reaction Conditions
5 cleavage sites
Digestion time with 1l of FastDigest ScaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp 65C, 10 min 16hours
FastDigest ScrFI (Bme1390I)

5...C 3...G
CN G G...3 G NC C...5 100l

DNA ( dcm ), 1.4% agarose
Formulation
184 cleavage sites
Methylation Effects
#FD1424
Reaction Conditions
Note
FastDigest ScrFI (Bme1390I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest ScrFI (Bme1390I) for complete digestion inactivation star activity Lambda DNA, 1g/20l Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 5 min 4 bp No 16hours
FastDigest SexAI (CsiI)

C W G G T...3 G G W C CA...5 #FD2114 50l
3...T DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...A C
Formulation
Methylation Effects
Dam, Dcm, CpG no effect. EcoBI, EcoKI: may overlap effect not determined.
Reaction Conditions
5 cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SexAI (CsiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 10 min 5 min 15 min 4 bp 65C, 5 min 16hours
61
1
FastDigest SfaNI (BmsI)

C A T C(N)5...3 G T A G(N)9...5 #FD2124 20l
Formulation
Methylation Effects
Reaction Conditions
Note
At least two copies of FastDigest SfaNI(BmsI) recognition site are required for efficient cleavage.
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SfaNI (BmsI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 16hours
FastDigest SfcI (BfmI)

3...G
R Y A G...3 A Y R TC...5 #FD1164 20l

DNA,1.0% agarose 38 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
5...CT
Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SfcI (BfmI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp 65C, 20 min 16hours
FastDigest SfiI
5...G 3...C
G C C N N N NN G G C C...3 C G G NN N N N C C G G...5 #FD1824 150l

pUC19-SfiI DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of pUC19 DNA with inserted SfiI recognition sites in 15min at 50C in 1XFastDigest Buffer.
Note
FastDigest SfiI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099). For cleavage with FastDigest SfiI at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (Wertzell, L.M. et al., J. Mol. Biol., 248, 581-595, 1995.)
Reaction Conditions
2 cleavage sites
Methylation Effects
Digestion time with 1l of FastDigest SfiI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 15 min 5 bp No 16hours
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62
1.
RESTRICTION ENZYMES
FastDigest SmaI
C CG G G...3 3...G G G C C C ...5 #FD0663 100l #FD0664 200l
5...C Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
DNA, 0.7% agarose
Methylation Effects
Reaction Conditions
3 cleavage sites
Digestion time with 1l of FastDigest SmaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp 65C, 5 min 16hours
FastDigest SnaBI (Eco105I)

5...T 3...A
A CG T A...3 T GC A T...5 #FD0404 50l

Formulation
Methylation Effects
Reaction Conditions
1 cleavage site
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SnaBI (Eco105I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 65C, 5 min 16hours
FastDigest SpeI (BcuI)

pUC19-BcuI DNA/Psp1406I, 1.0% agarose
T A G T...3 G A T CA...5 #FD1253 20l #FD1254 50l

3...T Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
5...AC
Formulation
1l of enzyme (1 FDU) cleaves 1g of pUC19 DNA with inserted BcuI recognition site/ Psp1406I fragments in 5min at 37C in 1XFastDigest Buffer. The control DNA is.
1 cleavage site
Methylation Effects
Reaction Conditions
Lambda, 1g/20l no cleavage sites
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SpeI (BcuI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 1 bp No 16hours
63
1
FastDigest SphI (PaeI)

5...G 3...C G
C A T GC...3 T A C G...5 20l

DNA, 0.7% agarose
Formulation
Methylation Effects
#FD0604
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SphI (PaeI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 5 bp 65C, 5 min 16hours
FastDigest SspI
5...A
A TA T T...3 T AT A A...5 #FD0774 100l

6 cleavage sites
Reaction Conditions
Formulation
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest SspI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 30 min 2 bp 65C, 5 min 1hour
FastDigest StuI (Eco147I)

5...A
G GC C T...3 C CG G A...5 #FD0424 100l

Formulation
Methylation Effects
Reaction Conditions
6 cleavage sites
Note
FastDigest StuI (Eco147I) is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest StuI (Eco147I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp 80C, 10 min 16hours
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RESTRICTION ENZYMES
FastDigest StyI (Eco130I)

W W G G...3 3...G G W W CC ...5 #FD0414 200l
DNA, 1.0% agarose 10 cleavage sites Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...CC
Formulation
Methylation Effects
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest StyI (Eco130I) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 20 min 20 min 3 bp 65C, 5 min 16hours
FastDigest SwaI (SmiI)

M13mp18 DNA/MvaI, 1.0% agarose
T T TA A A T...3 A A AT T T A...5 #FD1244 200l

5...A 3...T Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml
Formulation
1l of enzyme (1 FDU) cleaves 1g of M13mp18 DNA/MvaI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
1 cleavage site
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest SwaI (SmiI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 20 min 30 min 30 min 2 bp 65C, 15 min 1hour
FastDigest TaaI
5...A 3...T
C NG T...3 GN C A...5 20l

Formulation
Methylation Effects
#FD1364
Reaction Conditions
Digestion time with 1l of FastDigest TaaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 16hours
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FastDigest TaiI
5... 3... T
A C G T...3 G C A ...5 50l

Formulation
Methylation Effects
#FD1144
Reaction Conditions
Digestion time with 1l of FastDigest TaiI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp No 6hours
FastDigest TaqI
5...TC 3...A
400l
121 cleavage sites
G A...3 G CT...5 #FD0674
Formulation
Methylation Effects
Reaction Conditions
Note
FastDigest TaqI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
Digestion time with 1l of FastDigest TaqI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 4 bp No 16hours
FastDigest TatI
T A C W...3 C A T GW...5 #FD1294
3...W 5...WG
Formulation
20l
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest TatI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 15 min 15 min 20 min 5 bp No 1hour
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RESTRICTION ENZYMES
FastDigest TauI
5...G
C S GC...3 3...CG S C G...5 20l

Formulation
Note
FastDigest TauI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6X DNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
#FD1654
Reaction Conditions
Methylation Effects
Dam, Dcm, EcoBI, EcoKI no effect. CpG: completely overlaps blocked (p.178).
Digestion time with 1l of FastDigest TauI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 60 min 15 min 30 min 5 bp No 16hours
FastDigest TfiI (PfeI)

W T C...3 T W A G...5 #FD1784
3...C 5...G A
Formulation
50l
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: may overlap blocked (p.179). EcoBI: may overlap effect not determined.
Reaction Conditions
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest TfiI (PfeI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 10 min 5 min 2 bp 65C, 5 min 16hours
FastDigest Tru1I
3...A
A A ...3 A TT...5 #FD0984
5...TT
Formulation
50l
Methylation Effects
Reaction Conditions
Digestion time with 1l of FastDigest Tru1I bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 3 bp No 2hours
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FastDigest Tsp509I (TasI)

3...
A T T ...3 T T A A...5 #FD1354
5... A
Formulation
100l 1l of enzyme (1 FDU) cleaves 1g of pBR322 DNA in 5min at 65C in 1XFastDigest Buffer.
Methylation Effects
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest Tsp509I (TasI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 3 bp No 6hours
FastDigest TspRI (TscAI)

5... 3... N
N N C A S T G N N...3 N G T S A C N N ...5 100l

8 cleavage sites
Reaction Conditions
Formulation
Note
FastDigest TspRI (TscAI) produces DNA fragments with a 9-base 3-extension. This may result in atypical DNA band patterns. FastDigest TspRI (TscAI) may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns heat the digested DNA in the presence of 6XDNA Loading Dye & SDS solution (#R1151) or SDS prior to electrophoresis.
#FD2104
Reaction Conditions
Methylation Effects
bp from end of DNA required Thermal Incubation time without Digestion time with 1l of FastDigest TspRI (TscAI) for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 5 min 2 bp No 6hours
FastDigest XapI
A T T Y...3 T T A AR...5 #FD1383 50l #FD1384 100l
3...Y Both supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml 5...R A
Formulation
DNA, 0.7% agarose
Methylation Effects
Reaction Conditions
58 cleavage sites
Digestion time with 1l of FastDigest XapI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 4 bp 80C, 5 min 6hours
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RESTRICTION ENZYMES
FastDigest XbaI
T A G A...3 3...A G A T C T ...5 #FD0684 300l

Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml Supplied with: 10XFastDigest Buffer 2X1 ml 10XFastDigest Green Buffer 2X1 ml
5...TC
Formulation
1l of enzyme (1 FDU) cleaves 1g of lambda DNA dam/SmaI fragments in 5min at 37C in 1XFastDigest Buffer.
Methylation Effects
Reaction Conditions
1 cleavage site
Note
FastDigest XbaI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
#FD0685
750l
Digestion time with 1l of FastDigest XbaI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 2 bp 65C, 20 min 16hours
FastDigest XhoI
C G A G...3 A G C TC...5 #FD0694 400l
3...G DNA, 0.7% agarose Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml Supplied with: 10XFastDigest Buffer 3X1 ml 10XFastDigest Green Buffer 3X1 ml 5...CT
Formulation
Methylation Effects
Reaction Conditions
1 cleavage site
#FD0695
1200l
Digestion time with 1l of FastDigest XhoI bp from end of DNA required Thermal Incubation time without for complete digestion inactivation star activity Plasmid DNA, 1g/20l PCR product, ~0.2g/30l Genomic DNA, 1g/10l 5 min 5 min 10 min 2 bp 80C, 5 min 16hours
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Protocols and Recommendations

1.1. Fast DNA digestion
1. Prepare the reaction mixture at room temperature in the order indicated:
Volume Component Water, nuclease-free* (#R0581)
1.2. Reaction set-up for digestion of multiple DNA samples

1. Pipette 2l of each DNA* sample into labeled tubes. 2. Prepare a master mix for n+1 samples. Example of master mix (for 10 samples of plasmid DNA):
Water, nuclease-free* (#R0581) 10XFastDigest Buffer or 10XFastDigest Green Buffer FastDigest enzyme (10 + 1) x 15l = 165l (10 + 1) x 2l = 22l (10 + 1) x 1l = 11l
1.3. Double and multiple digestion of DNA

FastDigest enzymes allow simultaneous digestion of DNA with two or more enzymes in one digestion reaction. Use 1l of each enzyme and scale up the reaction conditions appropriately. The combined volume of all added enzymes should not exceed 1/10 of the total reaction volume. If enzymes require different incubation temperature perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage.
Plasmid DNA 15l
PCR product 17l 2l **
Genomic DNA 30l 5l 10l (5g) 5l 50l
10XFastDigest Buffer or 10XFastDigest 2l Green Buffer DNA* FastDigest enzyme Total volume
2l 10l (up to 1g) (~0.2g) 1l 20l 1l 30l
2. Mix gently and spin down. 3. Incubate at 37C in a heat block or water thermostat for 5-15 min.*** 4. Inactivate the enzyme (optional).***
3. Add 18l of master mix* into tubes containing DNA.
Note
* The volume of DNA can be scaled up to 10l or down to 0.5l depending on the DNA concentration. The volume of water and master mix should be corrected to keep the indicated total reaction volume.
Note
* The volume of water should be corrected to keep the indicated total reaction volume. The volume of DNA can be scaled up to 10l or down to 0.5l depending on the DNA concentration. ** Only 2l of 10XFastDigest buffer or 10XFastDigest Green Buffer is required for unpurified PCR product in a 30l reaction volume. *** See the Certificate of Analysis or Table1.3(p.71) for enzyme and substrate specific incubation time, recommended temperature and enzyme inactivation conditions. For fast simultaneous plasmid vector linearization and dephosphorylation protocol seep.338.
1.4. Scaling up DNA digestion reaction

DNA FastDigest enzyme 10XFastDigest Buffer or 10XFastDigest Green Buffer Total volume 1g 1l 2l 2g 2l 2l 3g 3l 3l 4g 4l 4l 5g 5l 5l
20l 20l 30l 40l 50l
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Reaction Conditions
Table 1.3. Reaction conditions for FastDigest restriction enzymes. FastDigest restriction enzyme FastDigest AatII FastDigest AccI (XmiI) FastDigest Acc65I FastDigest AciI (SsiI) FastDigest AclI (Psp1406I) FastDigest AcuI (Eco57I)* FastDigest AfeI (Eco47III) FastDigest AflII (BspTI) FastDigest AgeI (BshTI) FastDigest AjuI* FastDigest AleI (OliI) FastDigest AluI FastDigest Alw21I FastDigest Alw26I FastDigest AlwNI (CaiI) FastDigest ApaI FastDigest ApaLI (Alw44I) FastDigest AscI (SgsI) FastDigest AseI (VspI) FastDigest AsiSI (SfaAI) FastDigest AvaI (Eco88I) FastDigest AvaII (Eco47I) FastDigest AvrII (XmaJI) FastDigest BamHI FastDigest BanI (BshNI) FastDigest BbsI (BpiI) FastDigest BbvI (Lsp1109I) FastDigest BclI FastDigest BfaI (FspBI) FastDigest BglI FastDigest BglII FastDigest BlpI (Bpu1102I) FastDigest Bme1580I (BseSI) FastDigest BmtI (BspOI) FastDigest BplI* FastDigest BpmI (GsuI) FastDigest Bpu10I FastDigest BsaAI (Ppu21I) FastDigest BsaBI (BseJI) FastDigest BsaHI (Hin1I) FastDigest BsaJI (BseDI) FastDigest BseGI FastDigest BseNI FastDigest BseXI FastDigest Bsh1236I FastDigest BsiEI (Bsh1285I) FastDigest BsiWI (Pfl23II) FastDigest BslI (BseLI) FastDigest BsmBI (Esp3I)* FastDigest BsmFI (FaqI)* Specificity 53
GACGTC GTMKAC GGTACC CCGC(-3/-1) AACGTT CTGAAG(16/14) AGCGCT CTTAAG ACCGGT
RESTRICTION ENZYMES
Digestion time with 1l of Incubation bp from end of FastDigest enzyme, min time Reaction DNA required Thermal Lambda Plasmid PCR Genomic for complete inactivation without temp. star DNA DNA product DNA digestion activity 1g/20l 1g/20l ~0.2g/30l 1g/10l 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 37C 30C 37C 37C 65C 37C 37C 37C 65C 65C 37C 37C 37C 37C 37C 37C 15 5 5 5 5 15 5 5 5 5 5 15 5 5 5 5 5 5 5 no sites 5 5 5 5 5 5 5 5 5 5 5 5 5 5 15 15 15 5 5 5 5 5 5 15 5 15 15 5 5 15 20 5 5 5 5 30 5 5 5 5 5 15 5 5 5 5 60 5 5 5 5 5 10 5 60 5 5 5 15 5 20 5 5 5 20 30 15 5 5 5 5 5 5 15 5 15 15 5 5 15 15 60 5 5 5 15 5 5 5 10 10 15 5 5 5 20 10 5 5 60 5 5 5 5 5 5 5 15 5 5 30 5 5 15 30 15 30 5 5 5 5 5 5 15 5 15 15 5 5 15 15 5 5 5 5 15 5 5 5 5 5 15 5 5 5 10 5 5 30 5 5 5 10 5 15 5 10 5 20 5 20 5 30 5 30 15 15 5 5 5 5 5 5 15 5 15 15 5 5 15 5 2 3 2 3 2 4 4 3 ND 3 4 1 1 4 2 4 2 2 5 2 1 2 2 2 1 2 3 3 3 3 2 3 3 ND 2 3 1 3 1 3 2 2 3 1 3 3 2 1 3 80C, 5 min 65C, 5 min 65C, 5 min 65C, 5 min 65C, 5 min 65C, 5 min 65C, 5 min NO 80C, 5 min 65C, 5 min 65C, 5 min 65C, 5 min 80C, 20 min 65C, 5 min 65C, 10 min 65C, 5 min 80C, 5 min 65C, 20 min 65C, 5 min 80C, 5 min 65C, 5 min 80C, 20 min NO 80C, 5 min 65C, 10 min 65C, 10 min 65C, 5 min 80C, 20 min 80C, 5 min 65C, 5 min NO 80C, 5 min 80C, 15 min 80C, 5min 65C, 5 min 65C, 5 min 80C, 5 min 65C, 5 min NO 65C, 5 min 80C, 5 min 80C, 5 min 80C, 5 min 80C, 15 min 80C, 10 min 80C, 15 min 65C, 5 min NO 65C, 10 min 65C, 5 min 16 h 16 h 16 h 4h 16 h 16 h 16 h 16 h 16 h 16 h 6h 16 h 16 h 16 h 16 h 16 h 16h 16 h 16 h 6h 16 h 16 h 16 h 1h 16 h 16 h 1h 16 h 16 h 2h 16 h 16 h 16 h 1h 16 h 16 h 1h 6h 1h 16 h 16 h 16 h 16 h 16 h 16 h 1h 16 h 16 h 6h 16 h
(7/12)GAA(N)7TTGG(11/6)
CACNNNNGTG AGCT GWGCWC GTCTC(1/5) CAGNNNCTG GGGCCC GTGCAC GGCGCGCC ATTAAT GCGATCGC CYCGRG GGWCC CCTAGG GGATCC GGYRCC GAAGAC(2/6) GCAGC(8/12) TGATCA CTAG GCCNNNNNGGC AGATCT GCTNAGC GKGCMC GCTAGC
(8/13)GAG(N) 5CTC(13/8)
CTGGAG(16/14) CCTNAGC(-5/-2) YACGTR GATNNNNATC GRCGYC CCNNGG GGATG(2/0) ACTGG(1/-1) GCAGC(8/12) CGCG CGRYCG CGTACG CCNNNNNNNGG CGTCTC(1/5) GGGAC(10/14)
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Table 1.3.Reaction conditions for FastDigest restriction enzymes.
1
FastDigest restriction enzyme
Specificity 53
TTCGAA GGGCCC GDGCHC TGTACA CTCAG(10/8) TCATGA ACCTGC(4/8) CCGCTC(-3/-3) GCAATG(2/0) RCCGGY GCGCGC CCANNNNNNTGG GTATAC CCTNAGG ATCGAT GTAC CTNAG Gm6ATC TTTAAA CACNNNGTG GACNNNNNNGTC CGGCCG GACNNNNNGTC CTCTTC(1/4) GAGCTC GGTCTC(1/5) GGTNACC CCTNNNNNAGG RGGNCCY GAATTC GATATC GGCGCC GCNGC GGATG(9/13) TGCGCA RTGCGCAY RGCGCY GGCC GACGC(5/10) GCGC GTYRAC AAGCTT GANTC GCGC GTTAAC CCGG GTNNAC GCNNNNNNNGC GGTACC TCCGGA CGCGCGCG
FastDigest Bsp119I FastDigest Bsp120I FastDigest Bsp1286I (SduI) FastDigest Bsp1407I FastDigest BspCNI (BseMII)* FastDigest BspHI (PagI) FastDigest BspMI (BveI)* FastDigest BsrBI (MbiI) FastDigest BsrDI (BseMI) FastDigest BsrFI (Cfr10I) FastDigest BssHII (PteI) FastDigest BstXI FastDigest BstZ17I (Bst1107I) FastDigest Bsu36I (Eco81I) FastDigest ClaI (Bsu15I) FastDigest Csp6I FastDigest DdeI (HpyF3I) FastDigest DpnI

FastDigest DraI FastDigest DraIII (AdeI) FastDigest DrdI (AasI) FastDigest EagI (Eco52I) FastDigest Eam1105I FastDigest EarI (Eam1104I) FastDigest Ecl136II FastDigest Eco31I FastDigest Eco91I FastDigest EcoNI (XagI) FastDigest EcoO109I FastDigest EcoRI FastDigest EcoRV (Eco32I) FastDigest EheI FastDigest Fnu4HI (SatI) FastDigest FokI FastDigest FspI (NsbI) FastDigest FspAI FastDigest HaeII (BfoI) FastDigest HaeIII (BsuRI) FastDigest HgaI (CseI) FastDigest HhaI FastDigest HincII FastDigest HindIII FastDigest HinfI FastDigest HinP1I (Hin6I) FastDigest HpaI (KspAI) FastDigest HpaII FastDigest Hpy8I FastDigest HpyF10VI FastDigest KpnI FastDigest Kpn2I FastDigest MauBI
Digestion time with 1l of Incubation bp from end of FastDigest enzyme, min time Reaction DNA required Thermal Lambda Plasmid PCR Genomic for complete inactivation without temp. star DNA DNA product DNA digestion activity 1g/20l 1g/20l ~0.2g/30l 1g/10l 37C 5 5 5 5 1 80C, 5 min 16 h 37C 5 5 15 5 3 80C, 10 min 16 h 37C 5 5 5 5 2 80C, 15 min 1h 37C 5 5 5 5 3 80C, 10 min 16 h 55C 15 30 15 15 2 80C, 5 min 16 h 37C 5 10 5 10 3 80C, 5 min 0.5 h 37C 15 15 60 20 5 65C, 5 min 16 h 37C 5 5 5 5 3 65C, 5 min 16 h 55C 15 15 15 15 3 80C, 5 min 16 h 37C 5 5 10 5 2 NO 2h 37C 5 5 5 5 3 80C, 5 min 16 h 37C 5 5 5 5 4 80C, 5 min 0.5 h 37C 5 5 5 10 3 NO 6h 37C 5 5 5 5 2 80C, 10 min 16 h 37C 5 5 5 5 2 65C, 15 min 16 h 37C 5 5 5 5 2 80C, 10 min 16 h 37C 5 5 5 10 3 65C, 5 min 16 h not 37C ND 5 10 1 80C, 5 min 16 h applicable 37C 5 5 5 5 4 65C, 5 min 16 h 37C 5 5 5 5 2 80C, 5 min 6h 37C 5 5 5 5 1 80C, 10 min 1h 37C 5 20 20 5 3 65C, 5 min 16 h 37C 5 5 5 5 3 65C, 5 min 16 h 37C 5 5 5 5 3 80C, 5 min 6h 37C 5 5 5 5 1 65C, 5 min 6h 37C 5 5 5 5 3 65C, 5 min 16 h 37C 5 5 5 5 2 65C, 10 min 2h 37C 5 5 5 20 2 65C, 5 min 16 h 37C 5 10 15 5 2 65C, 5 min 16 h 37C 5 5 20 5 2 80C, 5 min 0.5 h 37C 5 5 5 5 2 NO 16 h 37C 5 5 5 5 2 65C, 5 min 6h 37C 5 5 10 5 3 65C, 5 min 16 h 37C 5 5 5 5 1 65C, 5 min 1h 37C 5 5 5 5 3 65C, 15 min 16 h 37C 5 5 5 5 4 65C, 5 min 16 h 37C 5 5 5 5 2 65C, 10 min 1h 37C 5 5 5 5 3 NO 16 h 37C 15 15 30 15 2 80C, 5 min 16 h 37C 5 5 5 5 1 NO 16 h 37C 5 5 5 10 1 65C, 5 min 16 h 37C 5 5 20 10 3 80C, 10 min 16 h 37C 5 5 5 5 2 65C, 20 min 16 h 37C 5 10 5 5 2 80C, 10 min 16 h 37C 5 5 5 5 3 65C, 20 min 0.5 h 37C 5 5 5 5 3 65C, 5 min 16 h 37C 5 5 5 10 2 80C, 5 min 16 h 37C 5 5 5 5 2 80C, 5 min 16 h 37C 5 5 5 5 3 80C, 5 min 16 h 37C 5 5 5 5 2 80C, 5 min 16 h 37C no sites 5 10 5 5 65C, 5 min 16 h
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RESTRICTION ENZYMES
FastDigest restriction enzyme FastDigest MboI FastDigest MboII FastDigest MfeI (MunI) FastDigest MluI FastDigest MlyI (SchI) FastDigest MnlI FastDigest MreI FastDigest MscI (MlsI) FastDigest MseI (SaqAI) FastDigest MslI (RseI) FastDigest MspI FastDigest MssI FastDigest MvaI FastDigest Mva1269I FastDigest NaeI (PdiI) FastDigest NciI (BcnI) FastDigest NcoI FastDigest NdeI FastDigest NheI FastDigest NlaIII (Hin1II) FastDigest NlaIV (BspLI) FastDigest NmuCI FastDigest NotI FastDigest NruI (RruI) FastDigest NsiI (Mph1103I) FastDigest NspI (XceI) FastDigest PacI FastDigest PdmI FastDigest PflMI (Van91I) FastDigest PfoI FastDigest PmlI (Eco72I) FastDigest PpuMI (Psp5II) FastDigest PshAI (BoxI) FastDigest PsiI (AanI) FastDigest PspFI FastDigest PstI FastDigest PsuI FastDigest PsyI FastDigest PvuI FastDigest PvuII FastDigest RsaI FastDigest RsrII (CpoI) FastDigest SacI FastDigest SalI FastDigest SanDI (KflI) FastDigest SapI (LguI) FastDigest Sau3AI (Bsp143I) FastDigest Sau96I (Cfr13I) FastDigest SbfI (SdaI) FastDigest ScaI FastDigest ScrFI (Bme1390I) FastDigest SexAI (CsiI)
Specificity 53 GATC
GAAGA(8/7) CAATTG ACGCGT GAGTC(5/5) CCTC(7/6) CGCCGGCG TGGCCA TTAA CAYNNNNRTG CCGG GTTTAAAC CCWGG GAATGC(1/-1) GCCGGC CCSGG CCATGG CATATG GCTAGC CATG GGNNCC
GTSAC
GCGGCCGC TCGCGA ATGCAT RCATGY TTAATTAA GAANNNNTTC CCANNNNNTGG TCCNGGA CACGTG RGGWCCY GACNNNNGTC TTATAA CCCAGC(-1/-5) CTGCAG RGATCY GACNNNGTC CGATCG CAGCTG GTAC CGGWCCG GAGCTC GTCGAC GGGWCCC GCTCTTC(1/4)
GATC
GGNCC CCTGCAGG AGTACT CCNGG ACCWGGT
Digestion time with 1l of Incubation bp from end of FastDigest enzyme, min time Reaction DNA required Thermal without Lambda Plasmid PCR Genomic temp. for complete inactivation star DNA DNA product DNA digestion activity 1g/20l 1g/20l ~0.2g/30l 1g/10l 37C 5 5 5 10 1 65C, 15 min 16 h 37C 5 5 5 5 2 65C, 5 min 16 h 37C 5 5 5 5 3 NO 16 h 37C 5 5 5 15 3 80C, 5 min 16 h 37C 5 5 30 5 2 80C, 5 min 1h 37C 5 5 5 5 2 65C, 5 min 16 h 37C no sites 5 5 5 3 80C, 5 min 16 h 37C 5 5 30 20 3 80C, 20 min 16 h 37C 5 5 5 5 4 65C, 5 min 16 h 37C 5 5 5 5 4 65C, 20 min 16 h 37C 5 5 5 5 3 NO 16 h 37C 5 5 5 5 2 65C, 10 min 16 h 37C 5 5 5 10 4 NO 1h 37C 5 5 5 5 3 65C, 5 min 16 h 37C ND 5 5 5 3 65C, 15 min 16 h 37C 5 5 5 5 3 80C, 20 min 16 h 37C 5 10 10 5 3 65C, 15 min 16 h 37C 5 5 60 30 3 65C, 5 min 6h 37C 5 15 5 5 5 65C, 5 min 6h 37C 5 10 5 10 4 80C, 5 min 16 h 37C 5 5 5 5 2 65C, 20 min 16 h 37C 5 5 5 10 2 65C, 5 min 16 h 37C no sites 30 5 10 2 80C, 5 min 16 h 37C 5 5 5 5 5 NO 4h 37C 5 5 5 10 3 65C, 15 min 6h 37C 5 5 5 5 2 65C, 5 min 16 h 37C no sites 5 5 5 2 65C, 10 min 16 h 37C 5 5 5 5 2 65C, 5 min 16 h 37C 5 5 5 5 3 65C, 10 min 6h 37C 5 5 5 5 3 65C, 5 min 16 h 37C 5 5 5 5 2 80C, 10 min 1h 37C 5 5 5 20 2 80C, 5 min 1h 37C 5 5 5 5 3 80C, 20 min 16 h 37C 5 5 5 5 5 65C, 5 min 16 h 37C 5 5 5 5 4 80C, 5 min 16 h 37C 5 5 30 5 3 NO 16 h 37C 5 5 5 5 2 80C, 5 min 16 h 37C 5 5 5 5 2 80C, 5 min 4h 37C 5 15 5 5 4 80C, 5 min 16 h 37C 5 5 5 5 1 NO 0.5 h 37C 5 5 5 5 3 NO 16 h 37C 5 5 5 5 1 65C, 5 min 16 h 37C 5 15 30 10 1 65C, 5 min 16 h 37C 5 5 60 5 3 65C, 10 min 16 h 37C 5 5 5 10 5 NO 16 h 37C 5 5 5 5 2 65C, 5 min 16 h 37C 5 5 10 5 4 65C, 20 min 16 h 37C 5 5 5 5 2 NO 16 h 37C 5 5 5 5 3 NO 1h 37C 5 5 5 5 4 65C, 10 min 16 h 37C 5 5 5 5 4 NO 16 h 37C 5 10 5 15 4 65C, 5 min 16 h
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FastDigest restriction enzyme FastDigest SfaNI (BmsI) FastDigest SfcI (BfmI) FastDigest SfiI FastDigest SmaI FastDigest SnaBI (Eco105I) FastDigest SpeI (BcuI) FastDigest SphI (PaeI) FastDigest SspI FastDigest StuI (Eco147I) FastDigest StyI (Eco130I) FastDigest SwaI (SmiI) FastDigest TaaI FastDigest TaiI FastDigest TaqI FastDigest TatI FastDigest TauI FastDigest TfiI (PfeI) FastDigest Tru1I FastDigest Tsp509I (TasI) FastDigest TspRI (TscAI) FastDigest XapI FastDigest XbaI FastDigest XhoI
Specificity 53
GCATC(5/9) CTRYAG GGCCNNNNNGGCC CCCGGG TACGTA ACTAGT GCATGC AATATT AGGCCT CCWWGG ATTTAAAT ACNGT ACGT TCGA WGTACW GCSGC GAWTC TTAA
AATT
CASTG(2/-7) RAATTY TCTAGA CTCGAG
Digestion time with 1l of Incubation bp from end of FastDigest enzyme, min time Reaction DNA required Thermal without Lambda Plasmid PCR Genomic temp. for complete inactivation star DNA DNA product DNA digestion activity 1g/20l 1g/20l ~0.2g/30l 1g/10l 37C 5 5 5 5 1 65C, 5 min 16 h 37C 5 5 5 5 2 65C, 20 min 16 h 50C no sites 15 15 15 5 NO 16 h 37C 5 5 5 5 1 65C, 5 min 16 h 37C 5 5 5 5 3 65C, 5 min 16 h 37C no sites 5 5 5 1 NO 16 h 37C 5 5 5 5 5 65C, 5 min 16 h 37C 5 5 5 30 2 65C, 5 min 1h 37C 5 5 5 5 3 80C, 10 min 16 h 37C 5 5 20 20 3 65C, 5 min 16 h 37C no sites 20 30 30 2 65C, 15 min 1h 65C 5 5 5 5 3 NO 16 h 65C 5 5 5 5 2 NO 6h 65C 5 5 5 5 4 NO 16 h 65C 15 15 15 20 5 NO 1h 55C 15 60 15 30 5 NO 16 h 37C 5 5 10 5 2 65C, 5 min 16 h 65C 5 5 5 10 3 NO 2h 65C 5 5 5 5 3 NO 6h 65C 5 5 5 5 2 NO 6h 37C 5 5 5 10 4 80C, 5 min 6h 37C 5 5 5 10 2 65C, 20 min 16 h 37C 5 5 5 10 2 80C, 5 min 16 h
Note
* for digestion with FastDigest BsmBI (Esp3I) add 1mM DTT. for digestion with FastDigest AcuI (Eco57I), FastDigest AjuI and FastDigest BspCNI (BseMII) add 0.01 mM SAM. for digestion with FastDigest BplI and FastDigest BsmFI (FaqI) add 0.05 mM SAM. for digestion with FastDigest BspMI (BveI) add 0.5M oligonucleotide. NO no thermal inactivation. Spin column purification or phenol/chloroform extraction and ethanol precipitation of DNA is recommended. ND not determined.
Single letter code

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1. CONVENTIONAL RESTRICTION ENZYMES
Conventional Restriction Enzymes

Fermentas conventional restriction endonucleases are classic restriction enzymes, which normally digest DNA in onehour and each require a specific reaction buffer for 100% activity.
Activity and Quality Control Assays

Activity Assay
One unit is the amount of enzyme required to hydrolyze 1g of substrate DNA in 60min in 50l of reaction mixture under the recommended conditions. To determine restriction enzyme activity, concentrated enzymes are first diluted to approximately 0,5-1units/l with an enzyme dilution buffer (20mM potassium phosphate (pH7.4), 200mM KCl, 1mMEDTA, 7mM 2-mercaptoethanol, 10% glycerol and 0.2mg/ml BSA) and then used to cleave DNA. In general, enzymes are assayed with phage DNA at 37C. However, some exceptions apply: Restriction enzymes that show optimum activity at temperatures other than 37C are assayed under their optimal temperature. Restriction enzymes without recognition sites on DNA are assayed with another specific DNA substrate. Restriction enzymes with only a few recognition sites on the DNA are assayed using DNA hydrolyzed with another restriction enzyme. Restriction enzymes sensitive to Dam or Dcm methylation are assayed with DNA (dam, dcm ).
Non-specific Nuclease and Crosscontamination Assay

Varying amounts of restriction enzyme (2-20units) are incubated for 16hours with 1g of substrate DNA under the recommended assay conditions. After electrophoretic separation of the DNA fragments, the characteristic banding patterns are examined for alterations. To pass the test, the restriction enzyme must yield an unaltered banding pattern under conditions of up to 160fold overdigestion (10 units x 16hours). For information regarding restriction enzyme star activity, see the product description or Certificate of Analysis supplied with each enzyme.
Blue/White (B/W) Cloning Assay

The Blue/White cloning assay is designed to test the integrity of DNA ends. pUC57 DNA is digested at unique sites within the lacZ reporter gene with 10 units of a restriction enzyme in its optimal buffer. After a 16hour incubation, the plasmid DNA is recircularized by ligation and transformed into E.coli XL1-Blue competent cells. The cells are then plated onto X-Gal/IPTG/ Amp agar. An intact lacZ gene will produce a blue colony. If the termini of the linearized pUC57 are altered by contaminating exodeoxynucleases, the lacZ reading frame is interrupted, which results in the appearance of white colonies. The higher the ratio of blue:white colonies, the higher the quality of the restriction enzyme. An enzyme conforms to this quality criterion if the number of white colonies does not exceed 3%. Details of the assay are given in the Certificate of Analysis of each product. The test is applicable for enzymes recognizing unique sites within the lacZ reporter gene, and for those lacking recognition sites in pUC57. In the latter case, the assay is performed with the mixture of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I DNA fragments representing three different types of termini (3-overhang, 5-overhang and blunt ends).
Ligation and Recleavage Assay

The ligation and re-cleavage assay tests the integrity of DNA ends. DNA fragments obtained after 2-, 10- and 50fold overdigestion (units/ g of DNA xhours) are ligated with T4 DNA ligase and then recut with the same restriction enzyme. Only DNA fragments with intact 5- and 3-termini are ligated by T4 DNA ligase, and only molecules with reconstructed recognition sites can then be cleaved by the same restriction enzyme. A restriction enzyme conforms to the quality criterion if the ligation efficiency of DNA fragments generated by digestion with the restriction enzyme doesnt depend on the fold of DNA overdigestion with the assayed enzyme. The percentage of DNA that can be successfully ligated and then re-cleaved is presented for each restriction enzyme both in its catalog description and the Certificate of Analysis supplied with each enzyme.
Quality Control Labeled Oligonucleotide (LO) Test

The labelled oligonucleotides are incubated with an excess of restriction enzyme, separated on a polyacrylamide gel under denaturing conditions and analyzed by phospho-imaging. The restriction enzyme passes this quality control test if there is no degradation of labelled oligonucleotides and no decrease in specific radioactivity (see Fig.1.1 on p.2).
Storage and Shipping

All conventional restriction enzymes should be stored at -20C. During shipment on dry ice, enzymes may freeze. This does not affect their quality all Fermentas enzymes are 100% active after at least three freeze-thaw cycles. For 24-48hour delivery, enzymes may be shipped on blue ice since their quality is not affected by short exposure to 4C.
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Product List and Cross-reference to FastDigest Restriction Enzymes

Table 1.4. Fermentas conventional restriction enzymes.
Conventional FastDigest restriction enzyme restriction enzyme AanI (PsiI) FastDigest PsiI (AanI)
AarI AasI (DrdI) AatII Acc65I (Asp718I) AdeI (DraIII) AjiI (BmgBI) AjuI AlfI AloI AluI Alw21I (BsiHKAI) Alw26I (BsmAI) Alw44I (ApaLI) ApaI BamHI BauI (BssSI) BclI BcnI (NciI) BcuI (SpeI) BdaI BfiI (BmrI) BfmI (SfcI) BfuI (BciVI) BglI BglII Bme1390I (ScrFI) BoxI (PshAI) BpiI (BbsI) BplI Bpu10I Bpu1102I (BlpI) BseDI (BsaJI) BseGI (BtsCI) BseJI (BsaBI) BseLI (BslI) BseMI (BsrDI) BseMII (BspCNI) BseNI (BsrI) BseSI (Bme1580I) BseXI (BbvI) Bsh1236I (BstUI) Bsh1285I (BsiEI) BshNI (BanI) BshTI (AgeI) Bsp68I (NruI) Bsp119I (BstBI) Bsp120I (PspOMI) Bsp143I (Sau3AI) Bsp1407I (BsrGI)
Prototype PsiI AarI DrdI AatII KpnI (GGTACC) DraIII BtrI AjuI AlfI AloI AluI HgiAI BsmAI ApaLI ApaI BamHI BsiI BclI CauII SpeI BdaI BfiI SfeI BciVI BglI BglII ScrFI PshAI BbvII BplI Bpu10I EspI SecI FokI (GGATG(9/13) ) BsaBI BsiYI BsrDI BseMII BsrI BseSI BbvI FnuDII McrI HgiCI AgeI NruI AsuII ApaI (GGGCCC) MboI Bsp1407I
Specificity 53 TTATAA CACCTGC(4/8) GACNNNNNNGTC GACGTC GGTACC CACNNNGTG CACGTC (7/12)GAA(N)7 TTGG(11/6) (10/12)GCA(N) 6TGC(12/10) (7/12-13)GAAC(N) 6TCC(12-13/7) AGCT GWGCWC GTCTC(1/5) GTGCAC GGGCCC GGATCC CACGAG(-5/-1) TGATCA CCSGG ACTAGT (10/12)TGA(N) 6TCA(12/10) ACTGGG(5/4) CTRYAG GTATCC(6/5) GCCNNNNNGGC AGATCT CCNGG GACNNNNGTC GAAGAC(2/6) (8/13)GAG(N) 5CTC(13/8) CCTNAGC(-5/-2) GCTNAGC CCNNGG GGATG(2/0) GATNNNNATC CCNNNNNNNGG GCAATG(2/0) CTCAG(10/8) ACTGG(1/-1) GKGCMC GCAGC(8/12) CGCG CGRYCG GGYRCC ACCGGT TCGCGA TTCGAA GGGCCC GATC TGTACA
Cat. #
ER2061 ER1581/2 ER1721 ER0991/2 ER0901/2 ER1231 ER1941 ER1951 ER1801 ER1491 ER0011/2 ER0021 ER0031 ER0041 ER1411/5 ER0051/2/3/5 ER1841 ER0721/2/5 ER0061 ER1251/2 ER1961 ER1591 ER1161/2 ER1501 ER0071/2 ER0081/2 ER1421/2 ER1431 ER1011/2 ER1311/2 ER1181 ER0091/2 ER1081/2 ER0871/2 ER1711 ER1201 ER1261/2 ER1401 ER0881/2 ER1441 ER1451/2 ER0921/2 ER0891 ER1001 ER1461/2 ER0111 ER0121 ER0131 ER0781/2 ER0931/2
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FastDigest DrdI (AasI) FastDigest AatII FastDigest Acc65I FastDigest DraIII (AdeI) FastDigest AjuI
FastDigest AluI FastDigest Alw21I FastDigest Alw26I FastDigest ApaLI (Alw44I) FastDigest ApaI FastDigest BamHI FastDigest BclI FastDigest NciI (BcnI) FastDigest SpeI (BcuI)
FastDigest SfcI (BfmI) FastDigest BglI FastDigest BglII FastDigest ScrFI (Bme1390I) FastDigest PshAI (BoxI) FastDigest BbsI (BpiI) FastDigest BpII FastDigest Bpu10l FastDigest BlpI (Bpu1102I) FastDigest BsaJI (BseDI) FastDigest BseGI FastDigest BsaBI (BseJI) FastDigest BslI (BseLI) FastDigest BsrDI (BseMI) FastDigest BspCNI (BseMII) FastDigest BseNI FastDigest Bme1580I (BseSI) FastDigest BseXI FastDigest Bsh1236I FastDigest BsiEI (Bsh1285I) FastDigest BanI (BshNI) FastDigest AgeI (BshTI) FastDigest NruI (RruI) FastDigest Bsp119I FastDigest Bsp120I FastDigest Sau3AI (Bsp143I) FastDigest Bsp1407I
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Table 1.4.Fermentas conventional restriction enzymes.
Conventional FastDigest restriction enzyme restriction enzyme BspLI (NlaIV) FastDigest NlaIV (BspLI) BspOI (BmtI) FastDigest BmtI (BspOI)
BspPI (AlwI) BspTI (AflII) Bst1107I (BstZ17I) BstXI Bsu15I (ClaI) BsuRI (HaeIII) BveI (BspMI) CaiI (AlwNI) CfrI (EaeI) Cfr9I (XmaI) Cfr10I (BsrFI) Cfr13I (Sau96I) Cfr42I (SacII) CpoI (RsrII) CseI (HgaI) Csp6I (CviQI) DpnI DraI Eam1104I (EarI) Eam1105I (AhdI) Ecl136II (EcoICRI) Eco24I (BanII) Eco31I (BsaI) Eco32I (EcoRV) Eco47I (AvaII) Eco47III (AfeI) Eco52I (EagI) Eco57I (AcuI) Eco57MI Eco72I (PmlI) Eco81I (Bsu36I) Eco88I (AvaI) Eco91I (BstEII) Eco105I (SnaBI) Eco130I (StyI) Eco147I (StuI) EcoO109I (DraII) EcoRI EcoRII EheI (SfoI) Esp3I (BsmBI) FaqI (BsmFI) FspAI FspBI (BfaI) GsuI (BpmI) HhaI Hin1I (BsaHI) Hin1II (NlaIII) Hin4I Hin6I (HinP1I)
Prototype NlaIV NheI (GCTAGC) BinI AflII SnaI BstXI ClaI HaeIII BspMI AlwNI CfrI SmaI (CCCGGG) Cfr10I AsuI SacII RsrII HgaI RsaI (GTAC) DpnI AhaIII Ksp632I Eam1105I SacI (GAGCTC) HgiJII Eco31I EcoRV AvaII Eco47III XmaIII Eco57I Eco57MI PmaCI SauI AvaI BstEII SnaBI StyI StuI DraII EcoRI EcoRII NarI (GGCGCC) Esp3I FinI FspAI MaeI GsuI HhaI AcyI NlaIII Hin4I HhaI (GCGC)
Specificity 53 GGNNCC GCTAGC GGATC(4/5) CTTAAG GTATAC CCANNNNNNTGG ATCGAT GGCC ACCTGC(4/8) CAGNNNCTG YGGCCR CCCGGG RCCGGY GGNCC CCGCGG CGGWCCG GACGC(5/10) GTAC Gm6ATC TTTAAA CTCTTC(1/4) GACNNNNNGTC GAGCTC GRGCYC GGTCTC(1/5) GATATC GGWCC AGCGCT CGGCCG CTGAAG(16/14) CTGRAG(16/14) CACGTG CCTNAGG CYCGRG GGTNACC TACGTA CCWWGG AGGCCT RGGNCCY GAATTC CCWGG GGCGCC CGTCTC(1/5) GGGAC(10/14) RTGCGCAY CTAG CTGGAG(16/14) GCGC GRCGYC CATG (8/13-14)GAY(N) 5VTC(13-14/8) GCGC
Cat. #
ER1151/2 ER2041 ER1321/2 ER0831 ER0701 ER1021/2 ER0141/2/5 ER0151 ER1741 ER1391 ER0161 ER0171/2 ER0181 ER0191 ER0201/2/5 ER0741/2 ER1901 ER0211 ER1701/2/5 ER0221/3 ER0231/2 ER0241 ER0251 ER0281 ER0291/2 ER0301/2/3/5 ER0311/2 ER0321/2 ER0331/2 ER0341/2 ER1671 ER0361 ER0371/2 ER0381 ER0391/2 ER0401/2 ER0411 ER0421/2 ER0261 ER0271/2/3/5 ER1921 ER0441 ER0451/2 ER1811 ER1661/2 ER1761/2 ER0461/2 ER1851 ER0471 ER1831 ER1601/2 ER0481
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FastDigest AflII (BspTI) FastDigest BstZ17I (Bst1107I) FastDigest BstXI FastDigest ClaI (Bsu15I) FastDigest HaeIII (BsuRI) FastDigest BspMI (BveI) FastDigest AlwNI (CaiI)
FastDigest BsrFI (Cfr10I) FastDigest Sau96I (Cfr13I) FastDigest RsrII (CpoI) FastDigest HgaI (CseI) FastDigest Csp6I FastDigest DpnI FastDigest DraI FastDigest EarI (Eam1104I) FastDigest Eam1105I FastDigest Ecl136II FastDigest Eco31I FastDigest EcoRV (Eco32I) FastDigest AvaII (Eco47I) FastDigest AfeI (Eco47III) FastDigest EagI (Eco52I) FastDigest AcuI (Eco57I) FastDigest PmlI (Eco72I) FastDigest Bsu36I (Eco81I) FastDigest AvaI (Eco88I) FastDigest Eco91I FastDigest SnaBI (Eco105I) FastDigest StyI (Eco130I) FastDigest StuI (Eco147I) FastDigest EcoO109I FastDigest EcoRI FastDigest EheI FastDigest BsmBI (Esp3I) FastDigest BsmFI (FaqI) FastDigest FspAI FastDigest BfaI (FspBI) FastDigest BpmI (GsuI) FastDigest HhaI FastDigest BsaHI (Hin1I) FastDigest NlaIII (Hin1II) FastDigest HinP1I (Hin6I)
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Conventional FastDigest restriction enzyme restriction enzyme HincII (HindII) FastDigest HincII HindIII FastDigest HindIII HinfI FastDigest HinfI HpaII FastDigest HpaII
HphI Hpy8I (MjaIV) HpyF3I (DdeI) HpyF10VI (MwoI) KpnI Kpn2I (BspEI) KspAI (HpaI) LguI (SapI) Lsp1109I (BbvI) LweI (SfaNI) MauBI MbiI (BsrBI) MboI MboII MlsI (MscI) MluI MnlI Mph1103I (NsiI) MreI (Sse232I) MspI (HpaII) MssI (PmeI) MunI (MfeI) MvaI (BstNI) Mva1269I (BsmI) NcoI NdeI NheI NmuCI (Tsp45I) NotI NsbI (FspI) OliI (AleI) PacI PaeI (SphI) PagI (BspHI) PasI PauI (BssHII) PdiI (NaeI) PdmI (XmnI) PfeI (TfiI) Pfl23II (BsiWI) PfoI PpiI Ppu21I (BsaAI) PscI (PciI) Psp5II (PpuMI) Psp1406I (AclI) PstI PsuI (BstYI)
Prototype HindII HindIII HinfI HpaII HphI MjaIV DdeI MwoI KpnI BspMII HpaI SapI BbvI SfaNI MauBI BsrBI MboI MboII BalI MluI MnlI AvaIII Sse232I HpaII PmeI MfeI EcoRII (CCWGG) BsmI NcoI NdeI NheI Tsp45I NotI MstI OliI PacI SphI BspHI PasI BsePI NaeI XmnI TfiI SplI PfoI PpiI BsaAI BspLU11I PpuMI AclI PstI XhoII
Specificity 53 GTYRAC AAGCTT GANTC CCGG GGTGA(8/7) GTNNAC CTNAG GCNNNNNNNGC GGTACC TCCGGA GTTAAC GCTCTTC(1/4) GCAGC(8/12) GCATC(5/9) CGCGCGCG CCGCTC(-3/-3) GATC GAAGA(8/7) TGGCCA ACGCGT CCTC(7/6) ATGCAT CGCCGGCG CCGG GTTTAAAC CAATTG CCWGG GAATGC(1/-1) CCATGG CATATG GCTAGC GTSAC GCGGCCGC TGCGCA CACNNNNGTG TTAATTAA GCATGC TCATGA CCCWGGG GCGCGC GCCGGC GAANNNNTTC GAWTC CGTACG TCCNGGA (7/12)GAAC(N) 5CTC(13/8) YACGTR ACATGT RGGWCCY AACGTT CTGCAG RGATCY
Cat. #
ER0491/2 ER0501/2/3/5 ER0801/2/3 ER0511/2 ER1101/2 ER1571/2 ER1881/2 ER1731/2 ER0521/2/3 ER0531/2 ER1031/2 ER1931/2 ER2071 ER1621/2 ER2081 ER1271 ER0811/2 ER0821/2 ER1211/2 ER0561/2 ER1071/2 ER0731/2 ER2021 ER0541/2 ER1341/2 ER0751/2 ER0551 ER0961/2 ER0571/2/5 ER0581/2/5 ER0971/2/5 ER1511 ER0591/2/3/5 ER1221 ER1631/2 ER2201 ER0601/2 ER1281/2 ER1861 ER1091/2 ER1521/2 ER1531/2 ER1781 ER0851 ER1751 ER1541 ER1971 ER1871/2 ER0761 ER0941/2 ER0611/2/5 ER1551
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FastDigest Hpy8I FastDigest DdeI (HpyF3I) FastDigest HpyF10VI FastDigest KpnI FastDigest Kpn2I FastDigest HpaI (KspAI) FastDigest SapI (LguI) FastDigest BbvI (Lsp1109I) FastDigest SfaNI (BmsI) FastDigest MauBI FastDigest BsrBI (MbiI) FastDigest MboI FastDigest MboII FastDigest MscI (MlsI) FastDigest MluI FastDigest MnlI FastDigest NsiI (Mph1103I) FastDigest MreI FastDigest MspI FastDigest MssI FastDigest MfeI (MunI) FastDigest MvaI FastDigest Mva1269I FastDigest NcoI FastDigest NdeI FastDigest NheI FastDigest NmuCI FastDigest NotI FastDigest FspI (NsbI) FastDigest AleI (OliI) FastDigest PacI FastDigest SphI (PaeI) FastDigest BspHI (PagI) FastDigest BssHII (PteI) FastDigest NaeI (PdiI) FastDigest PdmI FastDigest TfiI (PfeI) FastDigest BsiWI (Pfl23II) FastDigest PfoI FastDigest BsaAI (Ppu21I) FastDigest PpuMI (Psp5II) FastDigest AclI (Psp1406I) FastDigest PstI FastDigest PsuI
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Conventional FastDigest restriction enzyme restriction enzyme PsyI (Tth111I) FastDigest PsyI PvuI FastDigest PvuI PvuII FastDigest PvuII RsaI FastDigest RsaI RseI (MslI) FastDigest MslI (RseI) SacI FastDigest SacI SalI FastDigest SalI SatI (Fnu4HI) FastDigest Fnu4HI (SatI) ScaI FastDigest ScaI SchI (MlyI) FastDigest MlyI (SchI) SdaI (SbfI) FastDigest SbfI (SdaI) SduI (Bsp1286I) FastDigest Bsp1286I (SduI) SfaAI (AsiSI) FastDigest AsiSI (SfaAI) SfiI FastDigest SfiI
SgeI SgrDI SgsI (AscI) SmaI SmiI (SwaI) SmoI (SmlI) SmuI (FauI) SsiI (AciI) SspI SspDI (KasI) TaaI (HpyCH4III) TaiI (MaeII) TaqI TasI (Tsp509I) TatI TauI Tru1I (MseI) TscAI (TspRI) TsoI TstI Van91I (PflMI) VspI (AseI) XagI (EcoNI) XapI (ApoI) XbaI XceI (NspI) XhoI XmaJI (AvrII) XmiI (AccI) Nicking enzymes Nb.Bpu10I Nt.Bpu10I Nb.Mva1269I Homing enzyme I-SceI
Prototype Tth111I PvuI PvuII RsaI MslI SacI SalI Fnu4HI ScaI PleI (GAGTC(4/5) ) Sse8387I SduI SgfI SfiI SgeI SgrDI AscI SmaI SwaI SmlI FauI AciI SspI NarI (GGCGCC) Tsp4CI MaeII (ACGT) TaqI TspEI TatI TauI MseI TspRI TsoI TstI PflMI VspI EcoNI ApoI XbaI NspI XhoI AvrII AccI Nb.Bpu10I Nt.Bpu10I Nb.BsmI
Specificity 53 GACNNNGTC CGATCG CAGCTG GTAC CAYNNNNRTG GAGCTC GTCGAC GCNGC AGTACT GAGTC(5/5) CCTGCAGG GDGCHC GCGATCGC GGCCNNNNNGGCC m5CNNG(9/13) CGTCGACG GGCGCGCC CCCGGG ATTTAAAT CTYRAG CCCGC(4/6) CCGC(-3/-1) AATATT GGCGCC ACNGT ACGT TCGA AATT WGTACW GCSGC TTAA CASTG(2/-7) TARCCA(11/9) (8/13)CAC(N) 6TCC(12/7) CCANNNNNTGG ATTAAT CCTNNNNNAGG RAATTY TCTAGA RCATGY CTCGAG CCTAGG GTMKAC CCTNA GC GGANT CG CCTNAGC GG ANTCG GAATG C CTTACG TAGGG ATAACAGGGTAAT ATCCCTATT GTCCCATTA
Cat. #
ER1331 ER0621/2 ER0631/3/5 ER1121/2 ER2001/2 ER1131/2/5 ER0641/2/5 ER1641/2 ER0431/2 ER1371 ER1191/2 ER0651 ER2091 ER1821 ER2211 ER2031 ER1891/2 ER0661/2/3/5 ER1241 ER1981 ER1691 ER1791 ER0771/2 ER2191 ER1361/2 ER1141/2 ER0671/2 ER1351/2 ER1291 ER1651/2 ER0981/2/3 ER2101 ER1991 ER1911 ER0711/2 ER0911/2 ER1301 ER1381 ER0681/2/3/5 ER1471/2 ER0691/2/3/5 ER1561/2 ER1481/2 ER1681 ER2011 ER2051
Page
140 141 141 141 142 142 143 143 144 144 144 145 145 146 146 147 147 147 148 148 148 149 149 150 150 150 151 151 151 152 152 153 153 154 154 154 155 155 155 156 156 156 157 157 158 158
FastDigest AscI (SgsI) FastDigest SmaI FastDigest SwaI (SmiI)
FastDigest AciI (SsiI) FastDigest SspI FastDigest TaaI FastDigest TaiI FastDigest TaqI FastDigest Tsp509I (TasI) FastDigest TatI FastDigest TauI FastDigest Tru1I FastDigest TspRI (TscAI)
FastDigest PflMI (Van91I) FastDigest AseI (VspI) FastDigest EcoNI (XagI) FastDigest XapI FastDigest XbaI FastDigest NspI (XceI) FastDigest XhoI FastDigest AvrII (XmaJI) FastDigest AccI (XmiI)
I-SceI
ER1771
159
Alphabetic list of commercially available restriction enzymes see p.207.
79
1
Product Description
AanI (PsiI)
5...T 3...A
Activity in Five Buffer System, % B G O R Tango 2XTango 50-100 50-100 0-20 0-20 100 20-50
T AT A A ...3 A TA T T ...5 200 u

1ml
Concentration
10u/l
Ligation and Recleavage

After 50fold overdigestion with AanI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
#ER2061
Conditions for 100% Activity

1XBuffer Tango at 37C.
Supplied with: 10XBuffer Tango
Storage Buffer
AanI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 300mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest PsiI (AanI)
Digestion of Agarose-embedded DNA

Minimum 5units of the enzyme are required for complete digestion of 1g of agarose-embedded DNA in 16hours.
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR 0-20 0-20 NR 50-100
AarI
5...C 3...G
A C C T G C (N)4 ...3 T G G A C G (N)8 ...5 25 u

1ml 1ml 25l
Concentration
2u/l

After 10fold overdigestion with AarI, more than 95% of the DNA fragments can be ligated and recut.
#ER1581

1XBuffer AarI at 37C. Oligonucleotide 0.5M.
Supplied with: 10XBuffer AarI 10XBuffer Tango 50Xoligonucleotide Supplied with: 10XBuffer AarI 10XBuffer Tango 50Xoligonucleotide
Methylation Effects
Storage Buffer
AarI is supplied in: 10mMpotassiumphosphate (pH7.4 at25C), 100mMKCl, 1mMEDTA, 1mMDTT, 0.2mg/mlBSA and 50%(v/v)glycerol. DNAs, especially of those with a single AarI site. Still, a complete cleavage of some substrates with AarI is difficult to achieve. Greater than 10fold overdigestion with AarI may result in star activity. AarI may remain associated with the cleaved
#ER1582
125u
1ml 1ml 2x25l

Minimum 5units of the enzyme are required for digestion of 1g of agarose-embedded DNA in 16hours. DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Note
For cleavage with AarI at least two copies of its recognition sequence are required. Inclusion of 0.5M oligonucleotide with the AarI recognition sequence in the reaction mixture significantly improves cleavage of
AasI (DrdI)
A C N N N NN N G T C ...3 3...C T G N NN N N N C A G ...5
5...G
Activity in Five Buffer System, % B G O R Tango 2XTango 100 20-50 0-20 0-20 50-100* 0-20
* Star activity appears at a greater than 5fold overdigestion (5 u x 1 h).
Concentration
10u/l
Methylation Effects
#ER1721
300 u
1ml 1ml

1XBuffer B at 37C.
Supplied with: 10XBuffer B 10XBuffer Tango

Storage Buffer
AasI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 100mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest DrdI (AasI)
Note
Greater than 10fold overdigestion with AasI may result in star activity.

After 10fold overdigestion with AasI, more than 95% of the DNA fragments can be ligated and recut.
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80
AatII
5...G
A C G TC...3 3...CT G C A G ...5 300 u 1500 u

1ml
Concentration
10u/l
Methylation Effects
#ER0991 #ER0992


Both supplied with: 10XBuffer Tango
Storage Buffer
AatII is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest AatII
Note
Activity decreases if reaction buffer pHis not between 7.5 and 8.0 at 25C.

After 50fold overdigestion with AatII, more than 95% of the DNA fragments can be ligated and recut.
AccI AccII AccIII
Fermentas enzymes FastDigest AccI (XmiI) and XmiI (AccI) Fermentas enzymes FastDigest Bsh1236I and Bsh1236I (BstUI) Fermentas enzymes FastDigest Kpn2I and Kpn2I (BspEI) (different sensitivity to methylation)
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 20-50 100 20-50 20-50 50-100
Acc65I (Asp718I)
5...GG 3...C
T A C C...3 C A T GG...5 #ER0901 1000 u

Supplied with: 10XBuffer O 10XBuffer Tango Supplied with: 10XBuffer O 10XBuffer Tango 1ml 1ml
Concentration
10u/l
Methylation Effects

1XBuffer O at 37C.
Storage Buffer
Acc65I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

#ER0902
5000 u
2x1ml 1ml
Note
Acc65I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Also available as FastDigest Acc65I

After 50fold overdigestion with Acc65I, approximately 95% of the DNA fragments can be ligated and recut. Fermentas enzymes FastDigest PflMI (Van91I) and Van91I (PflMI) Fermentas enzymes FastDigest AciI (SsiI) and SsiI (AciI) Fermentas enzymes FastDigest AcII (Psp1406I) and Psp1406I (AclI) Fermentas enzymes FastDigest XapI and XapI (ApoI) Fermentas enzymes FastDigest AcuI (Eco57I) and Eco57I (AcuI) Fermentas enzymes FastDigest BsaHI (Hin1I) and Hin1I (BsaHI)
AccB7I AciI AclI AcsI AcuI AcyI
81
1
AdeI (DraIII)
A C N N NG T G...3 3...G T GN N N C A C...5
5...C
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 100 20-50 100 100* 20-50
Concentration
10u/l
Methylation Effects
Dam, Dcm no effect. CpG: may overlap cleavage impaired (p.179). EcoKI, EcoBI may overlap effect not determined.
#ER1231
500 u
1ml 1ml

1XBuffer G at 37C.
Supplied with: 10XBuffer G 10XBuffer Tango
Storage Buffer
AdeI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 300mM KCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Minimum 10 units of the enzyme are required for complete digestion of 1g of agaroseembedded DNA in 16hours.
Also available as FastDigest DraIII (AdeI)

After 10fold overdigestion with AdeI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Note
Greater than 20fold overdigestion with AdeI may result in star activity.
AfaI

Fermentas enzymes FastDigest Csp6I and Csp6I (CviQI) (different cleavage position); FastDigest RsaI and RsaI Fermentas enzymes FastDigest AfeI (Eco47III) and Eco47III (AfeI) Fermentas enzymes FastDigest AflII (BspTI) and BspTI (AflII) Fermentas enzymes FastDigest AgeI (BshTI) and BshTI (AgeI) Fermentas enzymes FastDigest DraI and DraI Fermentas enzymes FastDigest Eam1105I and Eam1105I (AhdI)
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR 20-50* NR NR 20-50*
AfeI AflII AgeI AhaIII AhdI
AjiI (BmgBI)
A CG T C...3 C A G...5 3...G T G
5...C
Concentration
5u/l
Methylation Effects
#ER1941
200 u
1ml 1ml

1XBuffer AjiI at 37C.
Supplied with: 10XBuffer AjiI 10XBuffer Tango
Storage Buffer
AjiI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Note
Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.

After 50fold overdigestion with AjiI, approximately 80% of the DNA fragments can be ligated. No more than 50% of these can be recut due to the asymmetric recognition sequence of AjiI. The remaining uncleaved ligation products may be cut by AatII and Eco72I (PmlI).
www.fermentas.com
82
AjuI
5...
Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM 0-20 50-100 20-50 100 50-100 50-100
(N)GAA(N)7TTGG(N)11 ...3 3...12(N)CTT(N)7AACC(N) 6 ...5

7
Concentration
5u/l

#ER1951
100 u
1ml 0.1ml 1ml

1XBuffer R at 37C. SAM 0.01mM.
Supplied with: 10XBuffer R 50XSAM 10XBufferTango
Note
AjuI requires Sadenosylmethionine for activity. Complete cleavage of some substrates with AjuI is difficult to achieve. AjuI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. AjuI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate.
Storage Buffer
AjuI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest AjuI

After 50fold overdigestion with AjuI, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation Effects
AleI
Fermentas enzymes FastDigest AleI (OliI) and OliI (AleI)

Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM 0-20 0-20 0-20 100 0-20 20-50
AlfI
5...10(N)GCA(N)6TGC(N)12 ...3 3...12(N)CGT(N)6ACG(N) 10...5
Concentration
2u/l
Methylation Effects
#ER1801
50 u
1ml 0.1ml 1ml

1XBuffer R at 37C. SAM 0.01mM.

Supplied with: 10XBuffer R 50XSAM 10XBufferTango
Storage Buffer
AlfI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 100mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
AlfI requires Sadenosylmethionine for activity. Complete cleavage of some substrates with AlfI is difficult to achieve. AlfI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.

After 20fold overdigestion with AlfI, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
83
1
AloI
5... 7(N)GAAC(N)6TCC(N)12-13...3 3...12-13(N)CTTG(N)6AGG(N)7
B 0-20
Activity in Five Buffer System, % G O R Tango 2XTango 0-20 0-20 100 20-50 100
Concentration
2u/l

After 10fold overdigestion with AloI, approximately 70% of the DNA fragments can be ligated and more than 80% of these can be recut.
...5
#ER1491
100 u
1ml 1ml

1XBuffer R at 30C.
Supplied with: 10XBuffer R 10XBuffer Tango
Storage Buffer
AloI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol. The presence of Sadenosylmethionine in the reaction mixture results in incomplete cleavage with AloI. Greater than 10fold overdigestion with AloI may result in star activity. AloI may remain associated with the cleaved DNA. This may cause DNA band shifting
Methylation Effects
Dam: may overlap effect not determined. Dcm, EcoKI, EcoBI no effect. CpG: may overlap cleavage impaired (p.179). during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Note
Incubation at 37C results in 20% activity. AloI produces double-strand cuts on both sides from the interrupted recognition site. Its unique feature is a degenerate cleavage point on the 3side of the recognition sequence (12 or 13 nt away).
AluI
5...A 3...T
B 50-100
Activity in Five Buffer System, % G O R Tango 2XTango 0-20 0-20 0-20 100 20-50
GC T...3 CG A...5 600 u

1ml
Concentration
10u/l

After 50fold overdigestion with AluI, approximately 95% of the DNA fragments can be ligated and recut.
#ER0011 #ER0012

Supplied with: 10XBuffer Tango Supplied with: 10XBuffer Tango
3000 u
2x1ml
Storage Buffer
AluI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.15%TritonX-100, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest AluI
AlwI
Fermentas enzyme BspPI (AlwI)

Activity in Five Buffer System, % G O R Tango 2XTango 20-50 100 50-100 20-50 50-100
Alw21I (BsiHKAI)
5...G 3...C W
B 0-20
W G C WC...3 C G W G...5 500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with Alw21I, more than 95% of the DNA fragments can be ligated and recut.
#ER0021

1XBuffer O at 37C.
Supplied with: 10XBuffer O 10XBuffer Tango
Storage Buffer
Alw21I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 300mMNaCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Alw21I
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84
Alw26I (BsmAI)
T C T C(N)1...3 3...C A G A G(N)5 ...5
5...G
B 50-100
Activity in Five Buffer System, % G O R Tango 2XTango 100 0-20 0-20 100 100
Concentration
10u/l

After 50fold overdigestion with Alw26I, more than 95% of the DNA fragments can be ligated and recut.
#ER0031
1000 u
1ml

Storage Buffer
Alw26I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Alw26I
Alw44I (ApaLI)
5...G T 3...C
B 50-100
Activity in Five Buffer System, % G O R Tango 2XTango 100 0-20 50-100 100 50-100
G C A C...3 A C G TG...5 1000 u

1ml
Concentration
10u/l

After 50fold overdigestion with Alw44I, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
#ER0041

Storage Buffer
Alw44I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest ApaLI (Alw44I)

AlwNI Aor13HI Aor51HI
Fermentas enzymes FastDigest AIwNI (CaiI) and CaiI (AlwNI) Fermentas enzymes FastDigest Kpn2I and Kpn2I (BspEI) Fermentas enzymes FastDigest AfeI (Eco47III) and Eco47III (AfeI)
Activity in Five Buffer System, % G O R Tango 2XTango 20-50 0-20 0-20 20-50 0-20
ApaI
5...G 3...C C
B 100
G G C CC...3 C G G G...5 3000 u 5000 u

2x1ml 1ml
Concentration
10u/l

#ER1411 #ER1415

1XBuffer B at 37C.
Both supplied with: 10XBuffer B 10XBuffer Tango
Storage Buffer
ApaI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 50mM NaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
Incubation at 30C results in a 2fold increase in activity. ApaI is inhibited by salt concentrations above 50 mM. ApaI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Also available as FastDigest ApaI

After 50fold overdigestion with ApaI, more than 95% of the DNA fragments can be ligated and recut.
Methylation Effects
85
1
ApaLI ApoI AscI AseI AsiSI AspI Asp700I Asp718I

Fermentas enzymes FastDigest ApaLI (Alw44I) and Alw44I (ApaLI) (different sensitivity to methylation) Fermentas enzymes FastDigest XapI and XapI (ApoI) Fermentas enzymes FastDigest AscI (SgsI) and SgsI (AscI) Fermentas enzymes FastDigest AseI (VspI) and VspI (AseI) Fermentas enzymes FastDigest AsiSI (SfaAI) and SfaAI (AsiSI) Fermentas enzymes FastDigest PsyI and PsyI (Tth111I) Fermentas enzymes FastDigest PdmI and PdmI (XmnI) Fermentas enzymes FastDigest Acc65I and Acc65I (Asp718I); FastDigest KpnI and KpnI (different cleavage position and different sensitivity to methylation) Fermentas enzymes FastDigest Eam1105I and Eam1105I (AhdI) Fermentas enzymes FastDigest Alw21I and Alw21I (BsiHKAI) Fermentas enzymes FastDigest Sau96I (Cfr13I) and Cfr13I (Sau96I) Fermentas enzymes FastDigest Bsp119I and Bsp119I (BstBI) Fermentas enzymes FastDigest AvaI (Eco88I) and Eco88I (AvaI) Fermentas enzymes FastDigest AvaII (Eco47I) and Eco47I (AvaII) Fermentas enzymes FastDigest NsiI (Mph1103I) and Mph1103I (NsiI) Fermentas enzymes FastDigest FspI (NsbI) and NsbI (FspI) Fermentas enzymes FastDigest AvrII (XmaJI) and XmaJI (AvrII) Fermentas enzymes FastDigest Bme1580I (BseSI) and BseSI (Bme1580I) Fermentas enzymes FastDigest MscI (MlsI) and MlsI (MscI)
AspEI AspHI AsuI AsuII AvaI AvaII AvaIII AviII AvrII BaeGI BalI
BamHI
5...G G
B 20-50*
A T C C...3 3...C C T A G G ...5 4000 u

2x1ml 1ml Supplied with: 10XBuffer BamHI 10XBuffer Tango Supplied with: 10XBuffer BamHI 10XBuffer Tango
Concentration
10u/l 50u/l, HC
Activity in Five Buffer System, % G O R Tango 2XTango 100 20-50 50-100* 100* 50-100
Methylation Effects
#ER0051

1XBuffer BamHI at 37C.

#ER0055
10000 u
4x1ml 1ml
Storage Buffer
BamHI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 200mM NaCl, 1mMDTT, 0.1mMEDTA, 0.15%TritonX-100, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
#ER0052 #ER0053
5x4000 u HC, 20000 u

4x1ml 1ml
Both supplied with: 10XBuffer BamHI 10XBuffer Tango

After 50fold overdigestion with BamHI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest BamHI
BanI BanII
www.fermentas.com
Fermentas enzymes FastDigest BanI (BshNI) and BshNI (BanI) Fermentas enzyme Eco24I (BanII)
86
BauI (BssSI)
5...CA
C G A G...3 3...G T G C TC...5 200 u

1ml Supplied with: 10XBuffer Tango
Concentration
10u/l

After 50fold overdigestion with BauI, more than 95% of the DNA fragments can be ligated and recut.
#ER1841

Methylation Effects
Storage Buffer
BauI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.15%TritonX-100, 0.2mg/mlBSA and 50%(v/v)glycerol.

BbeI

Fermentas enzymes FastDigest EheI and EheI (SfoI) (different cleavage position); Fermentas enzyme SspDI (KasI) (different cleavage position) Fermentas enzymes FastDigest PmlI (Eco72I) and Eco72I (PmlI) Fermentas enzymes FastDigest BbsI (BpiI) and BpiI (BbsI) Fermentas enzymes FastDigest SphI (PaeI) and PaeI (SphI) Fermentas enzymes FastDigest BbvI (Lsp1109I) and Lsp1109I (BbvI); FastDigest BseXI and BseXI (BbvI) Fermentas enzymes FastDigest BbsI (BpiI) and BpiI (BbsI) Fermentas enzyme BfuI (BciVI)
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 100 20-50 20-50 100* 100
BbrPI BbsI BbuI BbvI

BbvII BciVI
BclI
5...TG
A T C A...3 3...A C T A G T ...5 1000 u

1ml 1ml Supplied with: 10XBuffer G 10XBuffer Tango
Concentration
10u/l
Methylation Effects
#ER0721

1XBuffer G at 55C.
Storage Buffer
BclI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
Incubation at 37C results in 50% activity. Greater than 15fold overdigestion with BclI may result in star activity. BclI is blocked by dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099).
#ER0725 #ER0722
3000 u 5000 u
2x1ml 1ml
Both supplied with: 10XBuffer G 10XBuffer Tango

After 10fold overdigestion with BclI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest BclI
87
1
BcnI (NciI)
5...C 3...G
CS G G...3 G SC C...5 1000 u

1ml
Concentration
10u/l

After 10fold overdigestion with BcnI, more than 80% and more than 95% of these can be recut.
#ER0061

Methylation Effects
Storage Buffer
BcnI is supplied in: 10mM potassium phosphate (pH7.5 at 25C), 200mM NaCl, 1mMEDTA, 1mMDTT, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest NciI (BcnI)
BcuI (SpeI)
5...AC 3...T
T A G T...3 G A T CA...5 400 u 2000 u

1ml
Concentration
10u/l

After 50fold overdigestion with BcuI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
#ER1251 #ER1252

Storage Buffer
BcuI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 300mM KCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest SpeI (BcuI)

Minimum 10 units of the enzyme are required for complete digestion of 1g of agaroseembedded Adenovirus-2 DNA in 16hours.
Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM NR 100 0-20 20-50 50-100* 50-100
BdaI
5...10(N)TGA(N)6TCA(N)12 ...3 3...12(N)ACT(N)6AGT(N) 10...5
Concentration
2u/l
#ER1961
50 u
1ml 0.1ml 1ml

1XBuffer G at 30C. SAM 0.05mM.
Supplied with: 10XBuffer G 50XSAM 10XBufferTango
Storage Buffer
BdaI is supplied in: 10mM potassium phosphate (pH7.4 at 25C), 100mM NaCl, 1mMEDTA, 1mMDTT, 0.2mg/mlBSA and 50%(v/v)glycerol. BdaI produces double-strand cuts on both sides of the interrupted recognition site. In certain sequence contexts, the cleavage position may be shifted by one base pair. However, the cleavage position indicated above will predominate. Greater than 40fold overdigestion with BdaI may result in star activity. BdaI may remain associated with the cleaved
After 10fold overdigestion with BdaI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation Effects
Note
Incubation at 37C results in 30% activity. Requires S-adenosylmethionine for activity. Complete cleavage of some substrates with BdaI is difficult to achieve. BdaI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis. BdaI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
www.fermentas.com
88
1. CONVENTIONAL RESTRICTION ENZYMES BfaI

Fermentas enzymes FastDigest BfaI (FspBI) and FspBI (BfaI)
BfiI (BmrI)
5...A 3...T
1
C T G G G(N)5...3 G A C C C(N)4...5 50 u
1ml 0.5ml
Concentration
2u/l

After 10fold overdigestion with BfiI, more than 40% of the DNA fragments can be ligated and more than 90% of these can be recut.
#ER1591

Supplied with: 10XBuffer Tango 3XBfiI Stop Solution
Termination of Digestion Reaction

Do not attempt to stop the digestion reaction by adding EDTA (see Note). Use Stop Solution containing SDS or the supplied BfiI Stop Solution to terminate the reaction by incubating at 65C for 10 min. If the digested DNA is to be used in down-stream manipulations, inactivate BfiI by incubating at 65C for 20 min.
Methylation Effects
Note
BfiI restriction enzyme cleaves DNA specifically both in the presence and absence of Mg2+ ions in the reaction mixture (Sapranauskas, R., et al., J.Biol.Chem., 275, 30878-30885, 2000). Chelating of Mg2+ ions by EDTA does not inhibit BfiI activity and may cause nonspecific products. The non-specific cleavage increases at temperatures over 37C. Greater than 40fold overdigestion with BfiI may result in star activity.
3XBfiI Stop Solution (supplied)

0.6% SDS, 0.05% bromophenol blue and 50% glycerol.
Storage Buffer
BfiI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
BfmI (SfcI)
5...CT 3...G
R Y A G...3 A Y R TC...5 200 u 1000 u

1ml
Concentration
10u/l

After 50fold overdigestion with BfmI, more than 95% of the DNA fragments can be ligated and recut.
#ER1161 #ER1162

Methylation Effects
Storage Buffer
BfmI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest SfcI (BfmI)
Note
BfrI BfrBI
Fermentas enzymes FastDigest AflII (BspTI) and BspTI (AflII) Fermentas enzymes FastDigest NsiI (Mph1103I) and Mph1103I (NsiI) (different cleavage position)
89
1
BfuI (BciVI)
T A T C C(N)6...3 3...C A T A G G(N)5...5
5...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR 0-20 0-20 NR 50-100*

Concentration
5u/l
Methylation Effects
Dam, Dcm, EcoKI, EcoBI, CpG no effect.
#ER1501
100 u
1ml 1ml

1XBuffer BfuI at 37C.

Supplied with: 10XBuffer BfuI 10XBuffer Tango
Storage Buffer
BfuI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 300mMNaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
Greater than 5fold overdigestion with BfuI may result in star activity.

After 5fold overdigestion with BfuI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
BfuAI BfuCI

Fermentas enzymes FastDigest BspMI (BveI) and BveI (BspMI) Fermentas enzymes FastDigest Sau3AI (Bsp143I) and Bsp143I (Sau3AI); FastDigest DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest MboI and MboI (different sensitivity to methylation)
BglI
5...G 3...C
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 50-100 100 100 0-20 100
C C N N N NN G G C...3 G G NN N N N C C G...5 2000 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with BglI, more than 95% of the DNA fragments can be ligated and recut.
#ER0071

1XBuffer O at 37C.
Supplied with: 10XBuffer O 10XBuffer Tango Supplied with: 10XBuffer O 10XBuffer Tango
Storage Buffer
BglI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 300mMNaCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
#ER0072
5x2000 u
2x1ml 1ml

Also available as FastDigest BglI
BglII
5...A G 3...T
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 20-50 100 50-100 0-20 100
A T C T...3 C T A GA...5 500 u 2500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with BglII, more than 95% of the DNA fragments can be ligated and recut.
#ER0081 #ER0082

1XBuffer O at 37C.
Methylation Effects
Both supplied with: 10XBuffer O 10XBuffer Tango
Storage Buffer
BglII is supplied in: 10mMTris-HCl (pH8.2 at 25C), 200mM KCl, 1mMDTT, 0.1mMEDTA, 0.5mg/ml BSA and 50%(v/v)glycerol.

Also available as FastDigest BglII
www.fermentas.com
90
1. CONVENTIONAL RESTRICTION ENZYMES BinI BlnI BlpI

Fermentas enzyme BspPI (AlwI) Fermentas enzymes FastDigest AvrII (XmaJI) and XmaJI (AvrII) Fermentas enzymes FastDigest BlpI (Bpu1102I) and Bpu1102I (BlpI)
1
Bme1390I (ScrFI)
5...C 3...G
CN G G...3 G NC C...5 500 u 2500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with Bme1390I, more than 80% of the DNA fragments can be ligatedand more than 90% of these can be recut.
#ER1421 #ER1422

1XBuffer O at 37C.
Storage Buffer
Bme1390I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 200mM KCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Dam, EcoKI, EcoBI no effect. Dcm, CpG may overlap blocked (p.177, 179).
Also available as FastDigest ScrFI (Bme1390I)
Note
Bme1390I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Bme1580I BmgBI BmrI BmtI

Fermentas enzymes FastDigest Bme1580I (BseSI) and BseSI (Bme1580I) Fermentas enzyme AjiI (BmgBI) Fermentas enzyme BfiI (BmrI) Fermentas enzymes FastDigest BmtI (BspOI) and BspOI (BmtI); FastDigest NheI and NheI (different cleavage position) Fermentas enzymes FastDigest Bsp1286I (SduI) and SduI (Bsp1286I)
BmyI
BoxI (PshAI)
5...G 3...C
A C N NN N G T C...3 T G N NN N C A G...5 500 u

1ml
Concentration
10u/l
Methylation Effects
#ER1431

Storage Buffer
BoxI is supplied in: 10mM potassium phosphate (pH7.4 at 25C), 100mM NaCl, 1mMEDTA, 1mMDTT, 0.15%TritonX-100, 0.2mg/mlBSA and 50%(v/v) glycerol.

Also available as FastDigest PshAI (BoxI)
Note
Incubation at 30C results in a 2.5fold increase in activity.

After 50fold overdigestion with BoxI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
91
1
BpiI (BbsI)
5...G 3...C
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 100 50-100 50-100 50-100 50-100
A A G A C(N)2...3 T T C T G(N)6...5 200 u 1000 u

1ml 1ml
Concentration
10u/l
Methylation Effects
#ER1011 #ER1012

1XBuffer G at 37C.

Storage Buffer
BpiI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BbsI (BpiI)

After 50fold overdigestion with BpiI, more than 95% of the DNA fragments can be ligated and recut.
BplI
5... 8(N)G 3...13(N)C
Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM 0-20 20-50 0-20 0-20 100 20-50
A G(N)5C T C(N)13...3 T C(N)5G A G(N) 8...5 100 u

1ml 0.1ml
Concentration
5u/l

#ER1311

1XBuffer Tango at 37C. SAM 0.05mM.
Supplied with: 10XBuffer Tango 50XSAM Supplied with: 10XBuffer Tango 50XSAM
Note
BplI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.05mM S-adenosylmethionine gives more than a 100fold increase in BplI activity. Still, complete cleavage of some substrates with BplI is difficult to achieve. BplI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.
#ER1312
500 u
1ml 2x0.1ml
Storage Buffer
BplI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BpII

After 10fold overdigestion with BplI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation Effects
BpmI
Fermentas enzymes FastDigest BpmI (GsuI) and GsuI (BpmI)
www.fermentas.com
92
Bpu10I
CT N A G C...3 3...G G A N TC G ...5
5...C
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 20-50* 50-100* 100* 50-100* 100*
Concentration
5u/l
Methylation Effects
#ER1181
200 u
1ml 1ml

1XBuffer Bpu10I at 37C.
Note
For cleavage with Bpu10I at least two copies of its recognition sequence are required. A large excess of enzyme (>4 u/1g DNA) may result in incomplete DNA cleavage. Therefore, we recommend increasing the incubation time instead of using an excess of Bpu10I. Low salt, high glycerol (>5%) concentrations, pH>7.0 or a large excess of enzyme may result in star activity.
Supplied with: 10XBuffer Bpu10I 10XBuffer Tango
Storage Buffer
Bpu10I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 200mM KCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest Bpu10I

After 50fold overdigestion with Bpu10I, more than 90% of the DNA fragments can be ligated. No more than 50% of these can be recut due to asymmetric recognition sequence of Bpu10I.
Bpu1102I (BlpI)
5...G 3...C
CT N A G C...3 G A N TC G...5 200 u 1000 u

1ml
Concentration
10u/l
Methylation Effects
#ER0091 #ER0092


Storage Buffer
Bpu1102I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BlpI (Bpu1102I)

After 50fold overdigestion with Bpu1102I, more than 80% of the DNA fragments can be ligated. More than 95% of these can be recut.
BpuAI BsaI BsaAI BsaBI BsaHI BsaJI BsaMI BseAI
Fermentas enzymes FastDigest BbsI (BpiI) and BpiI (BbsI) Fermentas enzymes FastDigest Eco31I and Eco31I (BsaI) Fermentas enzymes FastDigest BsaAI (Ppu21I) and Ppu21I (BsaAI) Fermentas enzymes FastDigest BsaBI (BseJI) and BseJI (BsaBI) Fermentas enzymes FastDigest BsaHI (Hin1I) and Hin1I (BsaHI) Fermentas enzymes FastDigest BsaJI (BseDI) and BseDI (BsaJI) Fermentas enzymes FastDigest Mva1269I and Mva1269I (BsmI) Fermentas enzymes FastDigest Kpn2I and Kpn2I (BspEI)
93
1
BseDI (BsaJI)
5...CC 3...G
N N G G...3 G N N CC...5 300 u 1500 u

1ml
Concentration
10u/l

After 50fold overdigestion with BseDI, more than 95% of the DNA fragments can be ligated and recut.
#ER1081 #ER1082

Storage Buffer
BseDI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest BsaJI (BseDI)
Note
Incubation at 37C results in 10% activity.
BseGI (BtsCI)
5...G 3...C
G A T G N N...3 C T A CN N ...5 500 u 2500 u

1ml
Concentration
10u/l

After 50fold overdigestion with BseGI, more than 95% of the DNA fragments can be ligated and recut.
#ER0871 #ER0872

Storage Buffer
BseGI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mM NaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest BseGI
Note
BseJI (BsaBI)
5...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR 100* 100 NR NR 100*

A T N NN N A T C...3 3...C T A N N N N T A G...5 2000 u

1ml 1ml Supplied with: 10XBuffer O 10XBuffer Tango
Concentration
10u/l
Methylation Effects
Dam: may overlap blocked (p.176). Dcm, CpG, EcoKI no effect. EcoBI: may overlap effect not determined.
#ER1711

1XBuffer O at 65C.
Storage Buffer
BseJI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mM NaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
Incubation at 37C results in less than 10% activity. Greater than 20fold overdigestion with BseJI may result in star activity. BseJI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099).
Also available as FastDigest BsaBI (BseJI)

After 10fold overdigestion with BseJI, more than 95% of the DNA fragments can be ligated and recut.
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94
BseLI (BslI)
C N N N N NN N G G...3 3...G G N N N N N N N C C ...5
5...C
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 100 50-100 20-50 100 50-100
Concentration
10u/l

After 50fold overdigestion with BseLI, more than 95% of the DNA fragments can be ligated and recut.
#ER1201
500 u
1ml

Methylation Effects
Storage Buffer
BseLI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BslI (BseLI)
Note
Incubation at 37C results in 40% activity. BseLI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 20-50 0-20 100 50-100 50-100
BseMI (BsrDI)
5...G 3...C
C A A T G N N...3 G T T A CN N ...5 100 u 500 u

1ml 1ml
Concentration
5u/l

After 50fold overdigestion with BseMI, more than 95% of the DNA fragments can be ligated and approximately 80% of these can be recut.
#ER1261 #ER1262

1XBuffer R at 55C.
Both supplied with: 10XBuffer R 10XBuffer Tango
Storage Buffer
BseMI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest BsrDI (BseMI)
Note
BseMII (BspCNI)
5...C 3...G
T C A G(N)10...3 A G T C(N) 8...5 100 u

1ml 0.1ml
Concentration
1u/l
Methylation Effects
#ER1401

Note
Incubation at 37C results in 30% activity. Requires Sadenosylmethionine for activity. Sinefungin can replace SAM in the restriction reaction. In this case DNA is not methylated and more than 95% of the ligated BseMII fragments can be recut by this enzyme.
Supplied with: 10XBuffer Tango 50XSAM
Also available as FastDigest BspCNI (BseMII)
Storage Buffer
BseMII is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

After 10fold overdigestion with BseMII, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme (see Note).
95
1
BseNI (BsrI)
5...A 3...T
Activity in Five Buffer System, % B G O R Tango 2XTango 100 20-50 0-20 0-20 50-100 20-50
C T G G N...3 G A CC N ...5 1000 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with BseNI, more than 95% of the DNA fragments can be ligated and recut.
#ER0881

1XBuffer B at 65C.
Supplied with: 10XBuffer B 10XBuffer Tango Supplied with: 10XBuffer B 10XBuffer Tango
Storage Buffer
BseNI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
#ER0882
5000 u
2x1ml 1ml
Note
Incubation at 37C results in less than 10% activity.
Also available as FastDigest BseNI
BsePI
Fermentas enzymes FastDigest BssHII (PteI) and PauI (BssHII)
BseSI (Bme1580I)
5...G 3...CM
K G C MC...3 C G K G...5 500 u

1ml 1ml
Concentration
10u/l
Methylation Effects
Dam, Dcm, CpG, EcoBI no effect. EcoKI: may overlap cleavage impaired (p.181).
#ER1441

1XBuffer G at 55C.

Storage Buffer
BseSI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mM NaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest Bme1580I (BseSI)
Note
Incubation at 37C results in 20% activity. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.

After 50fold overdigestion with BseSI, more than 95% of the DNA fragments can be ligated and recut.
BseXI (BbvI)
5...G 3...C
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR NR NR NR NR
C A G C(N) 8...3 G T C G(N)12...5 100 u 500 u

1ml
Concentration
3u/l
Methylation Effects
#ER1451 #ER1452

1XBuffer BseXI at 65C.
Both supplied with: 10XBuffer BseXI
Storage Buffer
BseXI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 200mM NaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
Incubation at 37C results in 10% activity. Greater than 40fold overdigestion with BseXI may result in star activity. BseXI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Also available as FastDigest BseXI

After 10fold overdigestion with BseXI, more than 95% of the DNA fragments can be ligated recut.
www.fermentas.com
96
1. CONVENTIONAL RESTRICTION ENZYMES BseYI

Fermentas enzyme FastDigest PspFI (different cleavage position)
Bsh1236I (BstUI)
5...C 3...G
1
GC G...3 CG C...5 500 u 2500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with Bsh1236I, more than 95% of the DNA fragments can be ligated and recut.
#ER0921 #ER0922

1XBuffer R at 37C.
Storage Buffer
Bsh1236I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Bsh1236I
Bsh1285I (BsiEI)
5...C 3...G
G R YC G...3 CY R G C...5 600 u

1ml 1ml
Concentration
10u/l
Methylation Effects
Dam: may overlap cleavage impaired (p.176). Dcm, EcoKI, EcoBI no effect. CpG: completely overlaps blocked (p.178).
#ER0891

1XBuffer G at 37C.
Storage Buffer
Bsh1285I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 150mMKCl, 1mMDTT, 1mMEDTA, 0.15%TritonX-100, 0.5 mgml BSA and 50%(v/v)glycerol.

Also available as FastDigest BsiEI (Bsh1285I)
Note
Bsh1285I cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with Bsh1285I, more than 95% of the DNA fragments can be ligated and recut.
BshNI (BanI)
5...G G 3...C
Y R C C...3 C R Y GG...5 2000 u

1ml 1ml
Concentration
10u/l
Methylation Effects
Dam, EcoBI no effect. Dcm, CpG: may overlap cleavage impaired (p.177, 179). EcoKI: may overlap effect not determined.
#ER1001

1XBuffer O at 37C.
Storage Buffer
BshNI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 200mM KCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Also available as FastDigest BanI (BshNI)

After 50fold overdigestion with BshNI, more than 95% of the DNA fragments can be ligated and recut.
Note
BshNI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
97
1
BshTI (AgeI)
5...AC 3...T
C G G T...3 G G C CA...5 200 u 1000 u

1ml 1ml
Concentration
10u/l
Methylation Effects
#ER1461 #ER1462

1XBuffer O at 37C.
Storage Buffer
BshTI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Also available as FastDigest AgeI (BshTI)
Note

After 50fold overdigestion with BshTI, more than 95% of the DNA fragments can be ligated and recut.
BsiEI BsiHKAI BsiWI BsiYI BslI BsmI BsmAI BsmBI BsmFI BsoBI
Fermentas enzymes FastDigest BsiEI (Bsh1285I) and Bsh1285I (BsiEI) Fermentas enzymes FastDigest Alw21I and Alw21I (BsiHKAI) Fermentas enzymes FastDigest BsiWI (Pfl23II) and Pfl23II (BsiWI) Fermentas enzymes FastDigest BslI (BseLI) and BseLI (BslI) Fermentas enzymes FastDigest BslI (BseLI) and BseLI (BslI) Fermentas enzymes FastDigest Mva1269I and Mva1269I (BsmI) Fermentas enzymes FastDigest Alw26I and Alw26I (BsmAI) Fermentas enzymes FastDigest BsmBI (Esp3I) and Esp3I (BsmBI) Fermentas enzymes FastDigest BsmFI (FaqI) and FaqI (BsmFI) Fermentas enzymes FastDigest AvaI (Eco88I) and Eco88I (AvaI) (different sensitivity to methylation)
Bsp68I (NruI)
5...T 3...A
C GC G A...3 G CG C T...5 800 u

1ml 1ml
Concentration
10u/l
Methylation Effects
#ER0111

1XBuffer O at 37C.

Storage Buffer
Bsp68I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 200mM KCl, 1mMDTT, 0.1mMEDTA, 0.5mg/ml BSA and 50%(v/v)glycerol.
Also available as FastDigest NruI (RruI)
Note
Greater than 15fold overdigestion with Bsp68I may result in star activity.

After 10fold overdigestion with Bsp68I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
www.fermentas.com
98
Bsp119I (BstBI)
TC G A A...3 3...A A G CT T ...5
5...T
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 0-20 0-20 0-20 100 100
Concentration
10u/l
Methylation Effects
Dam, Dcm, EcoBI no effect. CpG: completely overlaps blocked (p.178). EcoKI: may overlap effect not determined.
#ER0121
2500 u
1ml

Storage Buffer
Bsp119I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Also available as FastDigest Bsp119I

After 50fold overdigestion with Bsp119I, more than 95% of the DNA fragments can be ligated and recut.
Bsp120I (PspOMI)
5...GG 3...C
G C C C...3 C C G GG...5 1500 u

1ml 1ml
Concentration
10u/l
Methylation Effects
#ER0131

1XBuffer B at 37C.

Storage Buffer
Note
Bsp120I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with Bsp120I, more than 95% of the DNA fragments can be ligated and recut.
Bsp143I (Sau3AI)
5...G 3...
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 20-50 0-20 0-20 50-100 20-50
A T C ...3 C T A G...5 300 u 1500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with Bsp143I more than 95% of the DNA fragments can be ligated and recut.
#ER0781 #ER0782

1XBuffer Bsp143I at 37C.
Both supplied with: 10XBuffer Bsp143I 10XBuffer Tango
Storage Buffer
Methylation Effects
Also available as FastDigest Sau3Al (Bsp143I)
Note
DpnI, Bsp143I and MboI all recognize the same sequence but have different methylation sensitivities (pp.175181) and cleavage sites.
Bsp1286I
Fermentas enzymes FastDigest Bsp1286I (SduI) and SduI (Bsp1286I)
99
1
Bsp1407I (BsrGI)
5...T G 3...A
T A C A...3 C A T GT...5 300 u 1500 u

1ml
Concentration
10u/l

After 50fold overdigestion with Bsp1407I more than 95% of the DNA fragments can be ligated and recut.
#ER0931 #ER0932

Storage Buffer
Methylation Effects

BspCNI BspDI BspEI BspHI
Fermentas enzymes FastDigest BspCNI (BseMII) and BseMII (BspCNI) Fermentas enzymes FastDigest ClaI (Bsu15I) and Bsu15I (ClaI) Fermentas enzymes FastDigest Kpn2I and Kpn2I (BspEI) Fermentas enzymes FastDigest BspHI (PagI) and PagI (BspHI)
BspLI (NlaIV)
5...G 3...C
G NN C C...3 C NN G G...5 200 u 1000 u

1ml
Concentration
10u/l

After 50fold overdigestion with BspLI, approximately 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
#ER1151 #ER1152

Storage Buffer
BspLI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 200mM NaCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest NlaIV (BspLI)
Note
BspLI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
BspLU11I BspMI BspMII
Fermentas enzyme PscI (PciI) Fermentas enzymes FastDigest BspMI (BveI) and BveI (BspMI) Fermentas enzymes FastDigest Kpn2I and Kpn2I (BspEI)
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 0-20 100 100 0-20 20-50
BspOI (BmtI)
5...G 3...C G
C T A GC...3 A T C G...5 200 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with BspOI, more than 95% of the DNA fragments can be ligated and recut.
#ER2041

1XBuffer O at 37C.
Storage Buffer
BspOI is supplied in: 10mMTris-HCl (pH8.2 at 25C), 500mMNaCl, 1mMDTT, 0.1mMEDTA, 0.15%TritonX-100, 0.5mg/ml BSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest BmtI (BspOI)

Minimum 20 units of the enzyme are required for complete digestion of 1g of agaroseembedded lambda DNA in 16hours.
www.fermentas.com
100
BspPI (AlwI)
G A T C(N)4...3 3...C C T A G(N)5 ...5
5...G
Concentration
2u/l
Methylation Effects
Dam: completely overlaps blocked (p.176). Dcm, CpG, EcoKI, EcoBI no effect.
#ER1321 #ER1322
100 u 500 u
1ml

Storage Buffer
BspPI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
Incubation at 37C results in 30% activity. BspPI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).

After 10fold overdigestion with BspPI, approximately 70% of the DNA fragments can be ligated and more than 50% of these can be recut.
BspQI
Fermentas enzymes FastDigest SapI (LguI) and LguI (SapI)
BspTI (AflII)
5...CT 3...G
T A A G...3 A A T TC...5 1000 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with BspTI, more than 95% of the DNA fragments can be ligated and more than 95% of these can be recut.
#ER0831

1XBuffer O at 37C.
Storage Buffer
BspTI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 200mM KCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest AflII (BspTI)

BspT104I BspT107I BsrI BsrBI BsrDI BsrFI BsrGI BsrSI BssHII BssKI BssSI Bst98I
Fermentas enzymes FastDigest Bsp119I and Bsp119I (BstBI) Fermentas enzymes FastDigest BanI (BshNI) and BshNI (BanI) Fermentas enzymes FastDigest BseNI and BseNI (BsrI) Fermentas enzymes FastDigest BsrBI (MbiI) and MbiI (BsrBI) Fermentas enzymes FastDigest BsrDI (BseMI) and BseMI (BsrDI) Fermentas enzymes FastDigest BsrFI (Cfr10I) and Cfr10I (BsrFI) Fermentas enzymes FastDigest Bsp1407I and Bsp1407I (BsrGI) Fermentas enzymes FastDigest BseNI and BseNI (BsrI) Fermentas enzyme FastDigest BssHII (PteI) and PauI (BssHII) Fermentas enzymes FastDigest ScrFI (Bme1390I) and Bme1390I (ScrFI) (different cleavage position) Fermentas enzyme BauI (BssSI) Fermentas enzymes FastDigest AflII (BspTI) and BspTI (AflII)
101
1
Bst1107I (BstZ17I)
5...G 3...C
T AT A C...3 A TA T G...5 500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with Bst1107I, more than 90% of the DNA fragments can be ligated and recut.
#ER0701

1XBuffer O at 37C.
Storage Buffer
Bst1107I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest BstZ17I (Bst1107I)

BstBI BstEII BstF5I BstNI BstOI BstPI BstUI
Fermentas enzymes FastDigest Bsp119I and Bsp119I (BstBI) Fermentas enzymes FastDigest Eco91I and Eco91I (BstEII) Fermentas enzymes FastDigest BseGI and BseGI (BtsCI); Fermentas enzyme FastDigest FokI (different cleavage position) Fermentas enzymes FastDigest MvaI and MvaI (BstNI); EcoRII (different cleavage position and different sensitivity to methylation) Fermentas enzymes FastDigest MvaI and MvaI (BstNI); EcoRII (different cleavage position and different sensitivity to methylation) Fermentas enzymes FastDigest Eco91I and Eco91I (BstEII) Fermentas enzymes FastDigest Bsh1236I and Bsh1236I (BstUI)
BstXI
5...C 3...G
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 100 100 50-100 50-100 100
C A N N N N NN T G G...3 G T NN N N N N A C C...5 500 u 2500 u

1ml 1ml
Concentration
10u/l
Methylation Effects
Dam, CpG, EcoKI, EcoBI no effect. Dcm: may overlap cleavage impaired (p.177).
#ER1021 #ER1022

1XBuffer O at 55C.

Storage Buffer
BstXI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BstXI
Note
Incubation at 37C results in 50% activity. Greater than 15fold overdigestion with BstXI may result in star activity. BstXI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with BstXI, approximately 95% of the DNA fragments can be ligated and recut.
BstYI BstZI BstZ17I
Fermentas enzymes FastDigest PsuI and PsuI (BstYI) Fermentas enzymes FastDigest EagI (Eco52I) and Eco52I (EagI) Fermentas enzymes FastDigest BstZ17I (Bst1107I) and Bst1107I (BstZ17I)
www.fermentas.com
102
Bsu15I (ClaI)
TC G A T...3 3...T A G CT A ...5
5...A
Concentration
10u/l
Methylation Effects
Dam: may overlap blocked (p.176). Dcm, EcoKI no effect. CpG: completely overlaps blocked (p.178). EcoBI: may overlap effect not determined.
#ER0141 #ER0145 #ER0142
600 u 1000 u
1ml

Both supplied with: 10XBuffer Tango Supplied with: 10XBuffer Tango
Storage Buffer
Bsu15I is supplied in: 10mMTris-HCl (pH8.0 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.5mg/ml BSA and 50%(v/v)glycerol.

3000 u
2x1ml
Also available as FastDigest ClaI (Bsu15I)

After 50fold overdigestion with Bsu15I, more than 95% of the DNA fragments can be ligated and recut.
Note
Bsu15I is blocked by overlapping dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099).
Bsu36I
Fermentas enzymes FastDigest Bsu36I (Eco81I) and Eco81I (Bsu36I)
BsuRI (HaeIII)
5...G 3...C
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 20-50 50-100 100 50-100 100
GC C...3 CG G...5 3000 u

2x1ml 1ml
Concentration
10u/l

After 50fold overdigestion with BsuRI, more than 95% of the DNA fragments can be ligated and recut.
#ER0151

1XBuffer R at 37C.
Storage Buffer
BsuRI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 50mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest HaeIII (BsuRI)
BtrI BtsCI
Fermentas enzyme AjiI (BmgBI) Fermentas enzymes FastDigest BseGI and BseGI (BtsCI); Fermentas enzyme FastDigest FokI (different cleavage position)
103
1
BveI (BspMI)
5...A 3...T
C C T G C (N)4 ...3 G G A C G (N)8 ...5 250 u

1ml 1ml 2X25l
Concentration
5u/l

After 50fold overdigestion with BveI, more than 90% of the DNA fragments can be ligated and recut.
#ER1741

1XBuffer O at 37C. Oligonucleotide 0.5M.
Supplied with: 10XBuffer O 10XBuffer Tango 50Xoligonucleotide
Methylation Effects
Storage Buffer
BveI is supplied in: 10mMTris-HCl (pH7.5 at25C), 150mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol. substrates with BveI is difficult to achieve. BveI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
Also available as FastDigest BspMI (BveI)
Note
At least two copies of BveI recognition site are required for efficient cleavage. Inclusion of 0.5M oligonucleotide with the BveI recognition sequence in the reaction mixture significantly improves cleavage of plasmid DNAs, especially of those with a single BveI site. Still, complete cleavage of some
BveI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
CaiI (AlwNI)
5...C 3...G
A G N N NC T G...3 T CN N N G A C...5 500 u

1ml
Concentration
10u/l
Methylation Effects
#ER1391

Storage Buffer
CaiI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Also available as FastDigest AlwNI (CaiI)
Note
CaiI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with CaiI, more than 95% of the DNA fragments can be ligated and recut.
CauII CelII CfoI

Fermentas enzymes FastDigest NciI (BcnI) and BcnI (NciI) Fermentas enzymes FastDigest BlpI (Bpu1102I) and Bpu1102I (BlpI) Fermentas enzymes FastDigest HinP1I (Hin6I) and Hin6I (HinP1I) (different cleavage position); FastDigest HhaI and HhaI
www.fermentas.com
104
CfrI (EaeI)
5...Y G
Activity in Five Buffer System, % B G O R Tango 2XTango 50-100* 50-100 0-20 0-20 100 0-20
G C C R...3 3...R C C G GY...5 200 u

1ml Supplied with: 10XBuffer Tango
Concentration
10u/l
Methylation Effects
#ER0161


Storage Buffer
CfrI is supplied in: 10mMTris-HCl (pH8.5 at 25C), 500mM KCl, 1mMDTT, 0.1mMEDTA, 0.5mg/ml BSA and 50%(v/v)glycerol.
Note
CfrI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with CfrI, more than 95% of the DNA fragments can be ligated and recut.
Cfr9I (XmaI)
5...CC 3...G
C G G G...3 G G C CC...5 300 u 1500 u

1ml 1ml
Concentration
10u/l
Methylation Effects
#ER0171 #ER0172

1XBuffer Cfr9I at 37C.
Both supplied with: 10XBuffer Cfr9I 10XBuffer Tango
Storage Buffer
Cfr9I is supplied in: 10mMTris-HCl (pH7.5 at 25C), 250mMKCl, 1mMDTT, 0.1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Note
To achieve complete digestion of substrate with Cfr9I, the concentration of DNA should be no less than 50g/ml in the reaction buffer.

After 50fold overdigestion with Cfr9I, more than 95% of the DNA fragments can be ligated and recut.
Cfr10I (BsrFI)
C G G Y...3 3...Y G G C CR...5 #ER0181 200 u
1ml 1ml Supplied with: 10XBuffer Cfr10I 10XBuffer Tango 5...RC
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 20-50 20-50 50-100* 20-50 50-100
Concentration
10u/l
Methylation Effects

1XBuffer Cfr10I at 37C.

Storage Buffer
Cfr10I is supplied in: 10mM potassium phosphate (pH7.4at25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BsrFI (Cfr10I)
Note
Greater than 5fold overdigestion with Cfr10I may result in star activity. For cleavage with Cfr10I at least two copies of its recognition sequence are required.
Ligation and Recutting

105
1
Cfr13I (Sau96I)
5...GG 3...C
N C C...3 C N GG...5 1000 u

1ml
Concentration
10u/l

#ER0191

Methylation Effects
Storage Buffer
Cfr13I is supplied in: 10mM potassium phosphate (pH7.4 at 25C), 100mMKCl, 1mMEDTA, 1mMDTT, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest Sau96I (Cfr13I)
Note
Cfr13I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).
Cfr42I (SacII)
5...C 3...G
C G CG G...3 GC G C C...5 1200 u 2000 u

1ml 1ml
Concentration
10u/l
Methylation Effects
#ER0201 #ER0205

1XBuffer B at 37C.

Both supplied with: 10XBuffer B 10XBuffer Tango Supplied with: 10XBuffer B 10XBuffer Tango
Storage Buffer
Cfr42I is supplied in: 10mM potassium phosphate (pH7.4 at 25C), 100mM NaCl, 1mMEDTA, 1mMDTT, 0.2mg/mlBSA and 50%(v/v)glycerol.
#ER0202
5x1200 u
2x1ml 1ml
Note
Certain sites in lambda DNA are difficult to cleave with Cfr42I, the same as with its prototype SacII. At least two copies of Cfr42I recognition site are required for efficient cleavage. Cfr42I activity is affected by high salt concentration. Trace amounts of sodium chloride remaining in the substrate DNA after completion of upstream applications may inhibit enzyme activity and result in impaired DNA cleavage.

ClaI
Fermentas enzymes FastDigest ClaI (Bsu15I) and Bsu15I (ClaI)
CpoI (RsrII)
5...C 3...G
GG W C C G...3 C C W GG C...5 200 u 1000 u

1ml
Concentration
10u/l

After 50fold overdigestion with CpoI, more than 95% of the DNA fragments can be ligated and recut.
#ER0741 #ER0742

Storage Buffer
CpoI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest RsrII (CpoI)
www.fermentas.com
106
CseI (HgaI)
5...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR 50-100* 50-100 100 100* 50-100
A C G C (N) 5...3 3...C T G C G (N)10 ...5 100 u

1ml 1ml Supplied with: 10XBuffer R 10XBuffer Tango
Concentration
5u/l
Methylation Effects
#ER1901

1XBuffer R at 37C.
Storage Buffer
CseI is supplied in: 10mMTris-HCl (pH7.5 at 25C), 100mMKCl, 1mMDTT, 0.1mMEDTA and 50%(v/v)glycerol.
Note
For cleavage with CseI at least two copies of its recognition sequence are required.
Also available as FastDigest HgaI (CseI)

After 50fold overdigestion with CseI, more than 95% of the DNA fragments can be ligated and recut.
CseI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
CspI
Fermentas enzymes FastDigest RsrII (CpoI) and CpoI (RsrII)
Csp6I (CviQI)
5...GT 3...C
A C...3 A TG...5 1500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with Csp6I, more than 95% of the DNA fragments can be ligated and recut.
#ER0211

1XBuffer B at 37C.
Storage Buffer
Csp6I is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMKCl, 1mMDTT, 1mMEDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Csp6I
Csp45I CviAII CviQI DdeI
Fermentas enzymes FastDigest Bsp119I and Bsp119I (BstBI) Fermentas enzymes FastDigest NlaIII (Hin1II) and Hin1II (NlaIII) (different cleavage position) Fermentas enzymes FastDigest Csp6I and Csp6I (CviQI) Fermentas enzymes FastDigest DdeI (HpyF3I) and HpyF3I (DdeI)
107
1
DpnI
5...G 3...C
Activity in Five Buffer System, % B G O R Tango 2XTango 100 100 50-100 50-100 100 50-100
m6A T C...3 Tm6A G...5 500 u 1000 u 2500 u

1 ml
Concentration
10u/l
Methylation Effects
Dam: does not cut dam DNA (p.176). Dcm, EcoKI, CpG no effect. EcoBI: may overlap effect not determined.
#ER1701 #ER1705 #ER1702

Storage Buffer
DpnI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Note
DpnI requires the presence of N6-methyladenine within the recognition sequence to cleave DNA. DNA purified from a dam+ strain will be a substrate for DpnI. DpnI will only cleave fully-adenomethylated dam sites. Hemi-adenomethylated dam sites DpnI cleaves 60Xmore slowly. DpnI, Bsp143I and MboI all recognize the same sequence but have different methylation sensitivities (pp.175181) and cleavage sites.
All supplied with: 10XBuffer Tango
Also available as FastDigest DpnI

After 50fold overdigestion with DpnI, more than 70% of the DNA fragments can be ligated and more than 95% of these can be recut.
DpnII
Fermentas enzymes FastDigest DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest Sau3AI (Bsp143I) and Bsp143I (Sau3AI) (different sensitivity to methylation); FastDigest MboI and MboI
DraI
5...T 3...A
T TA A A...3 A AT T T...5 1500 u

1 ml
Concentration
10u/l, 50u/l, HC

After 50fold overdigestion with DraI, more than 95% of the DNA fragments can be ligated and recut.
#ER0221 #ER0223

Methylation Effects
HC, 7500 u
2x1 ml
Storage Buffer
DraI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15%TritonX-100, 0.2mg/mlBSA and 50%(v/v) glycerol.
Also available as FastDigest DraI,

DraII DraIII DrdI EaeI EagI
Fermentas enzymes FastDigest Eco0109I and EcoO109I Fermentas enzymes FastDigest DraIII (AdeI) and AdeI (DraIII) Fermentas enzymes FastDigest DrdI (AasI) and AasI (DrdI) Fermentas enzyme CfrI (EaeI) Fermentas enzymes FastDigest EagI (Eco52I) and Eco52I (EagI)
www.fermentas.com
108
Eam1104I (EarI)
5...C
T C T T C(N)1...3 3...G A G A A G(N)4...5 300 u 1500 u

1 ml
Concentration
10u/l
Methylation Effects
#ER0231 #ER0232


Storage Buffer
Eam1104I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Also available as FastDigest EarI (Eam1104I)
Note

After 50fold overdigestion with Eam1104I, more than 95% of the DNA fragments can be ligated and recut.
Certain sites in DNA are difficult to cleave with Eam1104I, the same as with its prototype Ksp632I.
Eam1105I (AhdI)
5...G 3...C
A C N N NN N G T C...3 T G N NN N N C A G...5 1000 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER0241

1XBuffer Eam1105I at 37C.

Supplied with: 10XBuffer Eam1105I 10XBuffer Tango
Storage Buffer
Eam1105I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Also available as FastDigest Eam1105I
Note

After 10fold overdigestion with Eam1105I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
EarI
Fermentas enzymes FastDigest EarI (Eam1104I) and Eam1104I (EarI)
Ecl136II (EcoICRI)
5...G 3...C
A GC T C...3 T CG A G...5 1500 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with Ecl136II, more than 90% of the DNA fragments can be ligated and recut.
#ER0251

1XBuffer Ecl136II at 37C.
Supplied with: 10XBuffer Ecl136II 10XBuffer Tango
Storage Buffer
Ecl136II is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest Ecl136II

109
1
EclHKI EclXI
Fermentas enzymes FastDigest Eam1105I and Eam1105I (AhdI) Fermentas enzymes FastDigest EagI (Eco52I) and Eco52I (EagI)
Eco24I (BanII)
5...G 3...CY
R G C YC...3 C G R G...5 1500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with Eco24I, more than 95% of the DNA fragments can be ligated and recut.
#ER0281

Storage Buffer
Eco24I is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects

Eco31I (BsaI)
5...G 3...C
G T C T C(N)1...3 C A G A G(N)5...5 1000 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
Dam, EcoKI no effect. Dcm, CpG: may overlap cleavage impaired (177, 179). EcoBI: may overlap effect not determined.
#ER0291

1XBuffer G at 37C.
Supplied with: 10XBuffer G 10XBuffer Tango Supplied with: 10XBuffer G 10XBuffer Tango
Storage Buffer
#ER0292

5000 u
2x1 ml 1 ml
Also available as FastDigest Eco31I

After 50fold overdigestion with Eco31I, more than 95% of DNA fragments can be ligated and recut.
Note
Eco31I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099). Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
Eco32I (EcoRV)
5...G 3...C
A TA T C...3 T AT A G...5 2000 u

1ml 1ml
Concentration
10u/l 50u/l, HC

#ER0301

1XBuffer R at 37C.
Methylation Effects
#ER0305 #ER0302 #ER0303
4000 u 5x2000 u HC, 10000 u

2x1 ml 1 ml
Storage Buffer
Eco32I is supplied in: 25 mM Tris-HCl (pH7.5 at 25C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.

All supplied with: 10XBuffer R 10XBuffer Tango
Also available as FastDigest EcoRV (Eco32I)
www.fermentas.com
110
Eco47I (AvaII)
W C C...3 3...C C W GG ...5 #ER0311 800 u
1 ml 1 ml Supplied with: 10XBuffer R 10XBuffer Tango Supplied with: 10XBuffer R 10XBuffer Tango 5...GG
Concentration
10u/l


1XBuffer R at 37C.
Storage Buffer
Eco47I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
#ER0312
4000 u
2x1 ml 1 ml
Note
Eco47I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).
Also available as FastDigest AvaII (Eco47I)
Eco47III (AfeI)
5...A 3...T
G CG C T...3 C GC G A...5 200 u 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with Eco47III, more than 80% of DNA fragments can be ligated and more than 90% of these can be recut.
#ER0321 #ER0322

1XBuffer O at 37C.
Storage Buffer
Eco47III is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest AfeI (Eco47III)

Eco52I (EagI)
5...CG 3...G
G C C G...3 C C G GC...5 500 u 2500 u

1 ml 1 ml
Concentration
10u/l

#ER0331 #ER0332

1XBuffer Eco52I at 37C.
Both supplied with: 10XBuffer Eco52I 10XBuffer Tango
Storage Buffer
Eco52I is supplied in: 10 mM Tris-HCl (pH8.2 at 25C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest EagI (Eco52I)

Eco53kI
Fermentas enzymes FastDigest Ecl136II and Ecl136II (EcoICRI); Fermentas enzymes FastDigest SacI and SacI (different cleavage position)
111
1
Eco57I (AcuI)
5...C 3...G
Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM 100 100 20-50 20-50 50-100 50-100
T G A A G(N)16...3 A C T T C(N)14...5 200 u

1 ml 0.1 ml 1 ml
Concentration
5u/l

After 10fold overdigestion with Eco57I, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
#ER0341

1XBuffer G at 37C. SAM0.01mM.
Supplied with: 10XBuffer G 50XSAM 10XBuffer Tango Supplied with: 10XBuffer G 50XSAM 10XBuffer Tango
Storage Buffer
Eco57I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 50%(v/v) glycerol.
#ER0342
1000 u
1 ml 2x0.1 ml 1 ml
Methylation Effects
Also available as FastDigest AcuI (Eco57I)
Note
Eco57I requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.01mM S-adenosylmethionine gives a 100fold increase in Eco57I activity. Still, complete cleavage of some substrates with Eco57I is difficult to achieve. For cleavage with Eco57I at least two copies of its recognition sequence are required. Eco57I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity. Eco57I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Eco57MI
5...C 3...G
Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM 100 50-100 0-20 20-50 50-100 0-20
T G R A G(N)16...3 A C Y T C(N)14...5 50 u
1 ml 0.1 ml 1 ml
Concentration
2u/l

After 10fold overdigestion with Eco57MI, approximately 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
#ER1671

1XBuffer B at 37C. SAM 0.01mM.
Supplied with: 10XBuffer B 50XSAM 10XBuffer Tango
Storage Buffer
Eco57MI is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 50%(v/v) glycerol. Eco57MI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. Eco57MI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA
Methylation Effects
Dam, CpG, EcoKI no effect. Dcm: may overlap blocked (p.177). EcoBI: may overlap effect not determined. Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis. Eco57MI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Note
Eco57MI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.01mM S-adenosylmethionine gives more than a 100fold increase in Eco57MI activity. Still, complete cleavage of some substrates with Eco57MI is difficult to achieve.
www.fermentas.com
112
Eco72I (PmlI)
5...C
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR 0-20 0-20 100 20-50
A CG T G...3 3...G T G C A C ...5 2000 u

1 ml Supplied with: 10XBuffer Tango
Concentration
10u/l
Methylation Effects
#ER0361

Storage Buffer

Also available as FastDigest PmlI (Eco72I)
Note
Greater than 10fold overdigestion with Eco72I may result in star activity.

After 10fold overdigestion with Eco72I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Eco81I (Bsu36I)
5...C 3...G
CT N A G G...3 G A N TC C...5 500 u 2500 u

1 ml
Concentration
10u/l

After 10fold overdigestion with Eco81I, more than 80% of the DNA fragments can be ligated in a reaction mixture containing 20-40u of T4 DNA Ligase/1g of fragments and 10%PEG. More than 95% of these can be recut.
#ER0371 #ER0372

Storage Buffer
Also available as FastDigest Bsu36l (Eco81I)
Methylation Effects

Eco88I (AvaI)
5...CY 3...G
Activity in Five Buffer System, % B G O R Tango 2XTango 100 50-100 0-20 0-20 100 20-50
C G R G...3 R G C YC...5 1000 u

1 ml
Concentration
10u/l

#ER0381

Storage Buffer
Methylation Effects
Also available as FastDigest AvaI (Eco88I)

113
1
Eco91I (BstEII)
5...GG 3...C
T N A C C...3 C A N T GG...5 1000 u

1 ml 1 ml
Concentration
10u/l

#ER0391

1XBuffer O at 37C.
Storage Buffer
Methylation Effects
#ER0392
5000 u
2x1 ml 1 ml

Also available as FastDigest Eco91I
Eco105I (SnaBI)
5...T
Activity in Five Buffer System, % B G O R Tango 2XTango 100* 50-100 0-20 0-20 100 0-20
A CG T A...3 3...A T G C A T ...5 600 u

1 ml Supplied with: 10XBuffer Tango Supplied with: 10XBuffer Tango
Concentration
10u/l
Methylation Effects
#ER0401 #ER0402


3000 u
2x1 ml
Storage Buffer
Eco105I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.2mg/mlBSA and 50%(v/v) glycerol.
Also available as FastDigest SnaBI (Eco105I)
Note
Greater than 15fold overdigestion with Eco105I may result in star activity.

After 10fold overdigestion with Eco105I, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Eco130I (StyI)
5...CC 3...G
W W G G...3 G W W CC...5 2500 u

1 ml 1 ml
Concentration
10u/l

#ER0411

1XBuffer O at 37C.
Storage Buffer
Methylation Effects
Also available as FastDigest StyI (Eco130I)

www.fermentas.com
114
Eco147I (StuI)
5...A
G GC C T...3 3...T C C G G A ...5 1000 u

1 ml 1 ml Supplied with: 10XBuffer B 10XBuffer Tango Supplied with: 10XBuffer B 10XBuffer Tango
Concentration
10u/l
Methylation Effects
#ER0421

1XBuffer B at 37C.
Storage Buffer

#ER0422
5000 u
2x1 ml 1 ml
Note
Eco147I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).
Also available as FastDigest StuI (Eco147I)

After 50fold overdigestion with Eco147I, more than 90% of the DNA fragments can be ligated and recut. Fermentas enzymes FastDigest Ecl136II and Ecl136II (EcoICRI); FastDigest SacI and SacI (different cleavage position) Fermentas enzymes FastDigest EcoNI (XagI) and XagI (EcoNI) Fermentas enzymes FastDigest Eco91I and Eco91I (BstEII)
EcoICRI EcoNI EcoO65I
EcoO109I (DraII)
5...R 3...Y
GG N C C Y...3 C C N GG R...5 2000 u

1 ml
Concentration
10u/l
Methylation Effects
#ER0261

Storage Buffer
EcoO109I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50%(v/v) glycerol.

Minimum 5units of the enzyme is required for complete digestion of 1g of agarose-embedded DNA in 16hours.
Also available as FastDigest EcoO109I
Note
EcoO109I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with EcoO109I, more than 95% of the DNA fragments can be ligated and recut.
115
1
EcoRI
A T T C...3 3...C T T A AG ...5 #ER0271 5000 u
2x1 ml 1 ml Supplied with: 10XBuffer EcoRI 10XBuffer Tango Supplied with: 10XBuffer EcoRI 10XBuffer Tango 5...GA
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 NR 100 100* NR 100
Concentration
10u/l 50u/l, HC
Methylation Effects

1XBuffer EcoRI at 37C.

#ER0275
10000 u
4x1 ml 1 ml
Storage Buffer
EcoRI is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA, 0.15%TritonX-100 and 50%(v/v) glycerol.
Note
Low salt concentration, large excess of the enzyme, pH>8.0, or the replacement of Mg2+ by Mn2+ may result in star activity.
#ER0272 #ER0273
5x5000 u HC, 25000 u

5x1 ml 1 ml
All supplied with: 10XBuffer EcoRI 10XBuffer Tango

After 50fold overdigestion with EcoRI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest EcoRI
EcoRII
5...C 3...
C W G G ...3 G G W C C...5 200 u

1 ml 1 ml
Concentration
10u/l
Note
At least two copies of EcoRII recognition site are required for efficient cleavage. For cleavage of DNA substrates with only one copy of recognition site MvaI (#ER0551), neoschizomer of EcoRII, is recommended. EcoRII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis. EcoRII is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).
#ER1921

1XBuffer O at 37C.
Storage Buffer
EcoRII is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.

After 50fold overdigestion with EcoRII, more than 90% of the DNA fragments can be ligated and recut.
Methylation Effects
Dam, CpG, EcoKI, EcoBI no effect. Dcm: completely overlaps blocked (p.177).
EcoRV EcoT14I EcoT22I
Fermentas enzymes FastDigest EcoRV (Eco32I) and Eco32I (EcoRV) Fermentas enzymes FastDigest StyI (Eco130I) and Eco130I (StyI) Fermentas enzymes FastDigest NsiI (Mph1103I) and Mph1103I (NsiI)
www.fermentas.com
116
EheI (SfoI)
5...G
G CG C C...3 3...C C G C G G ...5 500 u

Concentration
10u/l
Methylation Effects
#ER0441


Storage Buffer
EheI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Also available as FastDigest EheI
Note

After 50fold overdigestion with EheI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
Unlike NarI, EheI completely digests and pBR322 DNAs. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
EspI
Fermentas enzymes FastDigest BlpI (Bpu1102I) and Bpu1102I (BlpI)
Esp3I (BsmBI)
5...C 3...G
Activity in Five Buffer System, % B+DTT G+DTT O+DTT R+DTT Tango+DTT 2XTango+DTT 100 20-50 0-20 0-20 100 0-20
G T C T C(N)1...3 C A G A G(N)5...5 200 u 1000 u

1 ml
Concentration
10u/l
Methylation Effects
#ER0451 #ER0452

1XBuffer Tango at 37C. DTT (#R0861/2) 1mM.

Storage Buffer
Esp3I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5mg/ml BSA and 50%(v/v) glycerol.
Also available as FastDigest BsmBI (Esp3I)
Note
The enzyme requires DTT (#R0861/2). Freshly made DTT should be added to the reaction buffer.

After 50fold overdigestion with Esp3I, more than 95% of the DNA fragments can be ligated and recut.
117
1
FaqI (BsmFI)
5...G 3...C
G G A C(N)10...3 C C T G(N)14...5 100 u

1 ml 0.1 ml
Concentration
2u/l
Note
FaqI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.05mM S-adenosylmethionine gives more than a 2fold increase in FaqI activity. Still, complete cleavage of some substrates is difficult to achieve. For cleavage with FaqI at least two copies of its recognition sequence are required. FaqI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. FaqI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
#ER1811

Supplied with: 10XBuffer Tango 50XSAM
Also available as FastDigest BsmFI (FaqI)
Storage Buffer
FaqI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.

After 10fold overdigestion with FaqI, more than 90% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
Methylation Effects
FatI FauI FbaI FinI FnuDII Fnu4HI FokI

Fermentas enzymes FastDigest NlaIII (Hin1II) and Hin1II (different cleavage position) Fermentas enzyme SmuI (FauI) Fermentas enzymes FastDigest BclI and BclI Fermentas enzymes FastDigest BsmFI (FaqI) and Faq (BsmFI) Fermentas enzymes FastDigest Bsh1236I and Bsh1236I (BstUI) Fermentas enzymes FastDigest Fnu4HI (SatI) and SatI (Fnu4HI) Fermentas enzymes FastDigest FokI; FastDigest BseGI and BseGI (BtsCI) (different cleavage position) Fermentas enzymes FastDigest FspI (NsbI) and NsbI (FspI)
FspI
FspAI
5...R 3...Y
T G CG C A Y...3 A C GC G T R...5 100 u 500 u

1 ml 1 ml
Concentration
5u/l

After 50fold overdigestion with FspAI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
#ER1661 #ER1662

1XBuffer O at 37C.
Storage Buffer
FspAI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest FspAI
Note
www.fermentas.com
118
FspBI (BfaI)
A G...3 3...G A TC ...5 #ER1761 #ER1762 500 u 2500 u
1 ml 5...CT
Concentration
10u/l

After 50fold overdigestion with FspBI, more than 80% of the DNA fragments can be ligated and more than 95% of these can be recut.

Storage Buffer
FspBI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest BfaI (FspBI)

GsaI
Fermentas enzyme FastDigest PspFI
GsuI (BpmI)
5...C 3...G
T G G A G(N)16...3 A C C T C(N)14...5 100 u 500 u

1 ml 1 ml
Concentration
5u/l
Note
Incubation at 37C results in 70% activity. GsuI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.01mM S-adenosylmethionine gives a 2fold increase in activity. GsuI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis. GsuI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
#ER0461 #ER0462

1XBuffer B at 30C.
Storage Buffer
GsuI is supplied in: 10 mM potassium phosphate (pH7.5 at 25C), 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50%(v/v) glycerol.
Also available as FastDigest BpmI (GsuI)

After 10fold overdigestion with GsuI, approximately 90% of the DNA fragments can be ligated and recut.
Methylation Effects
HaeII HaeIII HapII

Fermentas enzyme FastDigest HaeII (BfoI) Fermentas enzymes FastDigest HaeIII (BsuRI) and BsuRI (HaeIII) Fermentas enzymes FastDigest HpaII and HpaII; FastDigest MspI and MspI (HpaII) (different sensitivity to methylation) Fermentas enzymes FastDigest HgaI (CseI) and CseI (HgaI) Fermentas enzymes FastDigest Alw21I and Alw21I (BsiHKAI) Fermentas enzymes FastDigest BanI (BshNI) and BshNI (BanI) Fermentas enzyme Eco24I
HgaI HgiAI HgiCI HgiJII
119
1
HhaI
5...G 3...C G
C GC...3 C G...5 2000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with HhaI, more than 95% of the DNA fragments can be ligated and recut.
#ER1851

Storage Buffer
HhaI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest HhaI
Hin1I (BsaHI)
5...G 3...C
RC G Y C...3 Y G CR G...5 300 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
Dam, EcoKI no effect. Dcm: may overlap cleavage impaired (p.177). CpG: completely overlaps blocked (p.178). EcoBI: may overlap effect not determined.
#ER0471

1XBuffer G at 37C.
Storage Buffer
Hin1I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50%(v/v) glycerol.

Also available as FastDigest BsaHI (Hin1I)

After 50fold overdigestion with Hin1I, more than 95% of the DNA fragments can be ligated and recut.
Note
Hin1I cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).
Hin1II (NlaIII)
5... 3... G
C A T G...3 T A C ...5 300 u

1 ml 1 ml
Concentration
5u/l

After 50fold overdigestion with Hin1II, more than 95% of the DNA fragments can be ligated and recut.
#ER1831

1XBuffer G at 37C.
Storage Buffer
Hin1II is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.15%TritonX-100, 0.5mg/ml BSA and 50%(v/v) glycerol.
Methylation Effects
Also available as FastDigest NlaIII (Hin1II)
Note
More stable when stored at -70C. At -20C the half-life of Hin1II is 6 months.
www.fermentas.com
120
Hin4I
(N)GAY(N)5VTC(N)13-14...3 3... 13-14(N)CTR(N) 5BAG(N) 8 ...5 #ER1601 50 u
1 ml 0.1 ml Supplied with: 10XBuffer Tango 50XSAM
5...8
Concentration
2u/l

After 10fold overdigestion with Hin4I, more than 95% of the DNA fragments can be ligated and only 50% of these can be recut due to the methylation of the recognition sequence by restriction enzyme.

Storage Buffer
Hin4I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Dam: may overlap blocked (p.176). Dcm, EcoKI no effect. CpG: may overlap cleavage impaired (p.179). EcoBI: may overlap effect not determined. Its unique feature is a degenerate cleavage point on the 3 side of the recognition sequence (13 or 14 nt away). Hin4I is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).
Note
Hin4I requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.05mM S-adenosylmethionine gives more than a 10fold increase in Hin4I activity. Still, complete cleavage of some substrates with Hin4I is difficult to achieve.
Hin4I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. Hin4I produces double-strand cuts on both sides from the interrupted recognition site.
Hin6I (HinP1I)
5...G C 3...C
G C...3 G CG...5 2000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with Hin6I, more than 95% of the DNA fragments can be ligated and recut.
#ER0481

Storage Buffer
Hin6I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest HinP1I (Hin6I)
HinP1I

Fermentas enzymes FastDigest HhaI and HhaI (different cleavage position); FastDigest HinP1I (Hin6I) and Hin6I (HinP1I)
HincII (HindII)
5...G 3...C
T YR A C...3 A RY T G...5 500 u 2500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with HincII, more than 95% of the DNA fragments can be ligated and recut.
#ER0491 #ER0492

Storage Buffer
HincII is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5mg/ml BSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest HincII
HindII
Fermentas enzymes FastDigest HincII and HincII (HindII)
121
1
HindIII
5...AA 3...T
G C T T...3 T C G AA...5 5000 u

1 ml 1 ml
Concentration
10u/l 50u/l, HC
Methylation Effects
#ER0501
Supplied with: 10XBuffer R 10XBuffer Tango Supplied with: 10XBuffer R 10XBuffer Tango

1XBuffer R at 37C.

#ER0505
10000 u
4x1 ml 1 ml
Storage Buffer
HindIII is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
#ER0502 #ER0503
5x5000 u HC, 25000 u

2x1 ml 1 ml

After 50fold overdigestion with HindIII, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest HindIII
HinfI
5...G A 3...C
N T C...3 T N AG...5 2000 u

1 ml 1 ml
Concentration
10u/l 50u/l, HC

After 50fold overdigestion with HinfI, more than 95% of the DNA fragments can be ligated and recut.
#ER0801

1XBuffer R at 37C.
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: may overlap cleavage impaired (p.179). EcoBI: may overlap blocked (p.181).
#ER0802 #ER0803
5x2000 u HC, 10000 u

2x1 ml 1 ml
Storage Buffer
HinfI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
FastDigest HinfI
HpaI
Fermentas enzymes FastDigest HpaI (KspAI) and KspAI (HpaI)
HpaII
5...CC 3...G
G G...3 G CC...5 1000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with HpaII, more than 95% of the DNA fragments can be ligated and recut.
#ER0511 #ER0512

5000 u
2x1 ml
Storage Buffer
HpaII is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest HpaII
www.fermentas.com
122
HphI
5...G
G T G A(N)8...3 3...C C A C T(N)7...5 300 u 1500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
Dam, EcoBI: may overlap blocked (p.176, 181). Dcm, CpG, EcoKI no effect.
#ER1101 #ER1102

1XBuffer B at 37C.
Note
HphI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099). High glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
Storage Buffer
HphI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

After 50fold overdigestion with HphI, more than 70% of the DNA fragments can be ligated and more than 90% of these can be recut.
Hpy8I (MjaIV)
5...G 3...C
T NN A C...3 A NN T G...5 200 u 1000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with Hpy8I, more than 95% of the DNA fragments can be ligated and recut.
#ER1571 #ER1572

Storage Buffer
Hpy8I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Hpy8I
HpyCH4III HpyCH4IV
Fermentas enzymes FastDigest TaaI and TaaI (HpyCH4III) Fermentas enzymes FastDigest TaiI and TaiI (MaeII) (different cleavage position)
HpyF3I (DdeI)
5...C T 3...G
N A G...3 A N TC...5 500 u 2500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with HpyF3I, more than 95% of the DNA fragments can be ligated and recut.
#ER1881 #ER1882

Storage Buffer
HpyF3I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest DdeI (HpyF3I)
123
1
HpyF10VI (MwoI)
5...G 3...C
C N N N N NN N G C...3 G N NN N N N N C G...5 300 u 1500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with HpyF10VI, more than 95% of the DNA fragments can be ligated and recut.
#ER1731 #ER1732

Storage Buffer
HpyF10VI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest HpyF10VI
Hsp92I Hsp92II ItaI KasI

Fermentas enzymes FastDigest BsaHI (Hin1I) and Hin1I (BsaHI) Fermentas enzymes FastDigest NlaIII (Hin1II) and Hin1II (NlaIII) Fermentas enzymes FastDigest Fnu4HI (SatI) and SatI (Fnu4HI) Fermentas enzymes FastDigest EheI and EheI (SfoI) (different cleavage position) Fermentas enzyme SspDI (KasI)
KpnI
5...G 3...CC
G T A CC...3 A T G G...5 4000 u

2x1 ml 1 ml
Concentration
10u/l 50u/l, HC

After 50fold overdigestion with KpnI, more than 95% of the DNA fragments can be ligated and recut.
#ER0521
Supplied with: 10XBuffer KpnI 10XBuffer Tango

1XBuffer KpnI at 37C.
Methylation Effects
#ER0522 #ER0523
5x4000 u HC, 20000 u

4x1 ml 1 ml
Storage Buffer
KpnI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Both supplied with: 10XBuffer KpnI 10XBuffer Tango
Note
High glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
Also available as FastDigest KpnI
Kpn2I (BspEI)
5...TC 3...A
C G G A...3 G G C CT...5 500 u 2500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with Kpn2I, more than 95% of the DNA fragments can be ligated and recut.
#ER0531 #ER0532

Storage Buffer
Kpn2I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Kpn2I

Note
www.fermentas.com
124
1. CONVENTIONAL RESTRICTION ENZYMES KspI Ksp632I

Fermentas enzyme Cfr42I (SacII) Fermentas enzymes FastDigest EarI (Eam1104I) and Eam1104I (EarI)
1
Activity in Five Buffer System, % B G O R Tango 2XTango 100 50-100* 20-50 20-50 100* 50-100
KspAI (HpaI)
5...G
T TA A C...3 3...C A A T T G ...5 500 u 2500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
Dam, Dcm, EcoBI no effect. CpG: may overlap cleavage impaired (p.179). EcoKI: may overlap blocked (p.181).
#ER1031 #ER1032

1XBuffer B at 37C.
Storage Buffer
KspAI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.

Also available as FastDigest HpaI (KspAI)
Note
High glycerol (>5%) concentration, pH>8.0 or a large excess of enzyme may result in star activity.

After 50fold overdigestion with KspAI, more than 90% of the DNA fragments can be ligated and recut.
LguI (SapI)
5...G 3...C
C T C T T C(N)1...3 G A G A A G(N)4...5 100 u 500 u

1 ml
Concentration
5u/l
Methylation Effects
#ER1931 #ER1932


Storage Buffer
LguI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest SapI (LguI)
Note

After 50fold overdigestion with LguI, more than 90% of the DNA fragments can be ligated and recut.
Lsp1109I (BbvI)
5...G
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 20-50* 50-100* 100* 20-50* 20-50*
C A G C(N) 8...3 3...C G T C G(N)12 ...5 200 u

1 ml 1 ml Supplied with: 10XBuffer Lsp1109I 10XBuffer Tango
Concentration
5u/l
Methylation Effects
#ER2071

1XBuffer Lsp1109I at 37C.
Note
Greater than 10fold overdigestion with Lsp1109I may result in star activity. Lsp1109I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis. Protocols and Recommendations p.160
Storage Buffer
Lsp1109I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BbvI (Lsp1109I)

After 10fold overdigestion with Lsp1109I, more than 95% of the DNA fragments can be ligated and recut.
125
1
LweI (SfaNI)
5...G 3...C
C A T C(N)5...3 G T A G(N)9...5 100 u 500 u

1 ml
Concentration
10u/l
Methylation Effects
#ER1621 #ER1622

Storage Buffer
LweI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
At least two copies of LweI recognition site are required for efficient cleavage. LweI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Also available as FastDigest SfaNI (BmsI)

After 50fold overdigestion with LweI, more than 95% of the DNA fragments can be ligated and recut.
MaeI MaeII MamI
Fermentas enzymes FastDigest BfaI (FspBI) and FspBI (BfaI) Fermentas enzymes FastDigest TaiI and TaiI (MaeII) (different cleavage position) Fermentas enzymes FastDigest BsaBI (BseJI) and BseJI (BsaBI)
MauBI
5...C 3...G
GC G C G C G...3 C G C G CG C...5 100 u

1 ml 1 ml
Concentration
5u/l
Methylation Effects
#ER2081

Supplied with: 10XBuffer Tango 10XBuffer Tango

Storage Buffer
MauBI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest MauBI

After 10fold overdigestion with MauBI, more than 90% of the DNA fragments can be ligated and recut.
MbiI (BsrBI)
5...C 3...G
C GC T C...3 G CG A G...5 1000 u

1 ml
Concentration
10u/l
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: completely overlaps cleavage impaired (p.178). EcoBI: may overlap effect not determined.
#ER1271

Storage Buffer
MbiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest BsrBI (MbiI)


After 50fold overdigestion with MbiI, approximately 80% of the DNA fragments can be ligated. No more than 50% of these can be recut due to the asymmetric recognition sequence of MbiI.
Note
www.fermentas.com
126
MboI
5...G 3...
A T C ...3 C T A G...5 300 u 1500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER0811 #ER0812

1XBuffer R at 37C.
Storage Buffer
MboI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
MboI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099). MboI, Bsp143I and DpnI all recognize the same sequence but have different methylation sensitivities (pp.175181) and cleavage sites.
Also available as FastDigest MboI

After 50fold overdigestion with MboI, more than 95% of the DNA fragments can be ligated and recut.
MboII
5...G 3...C
A A G A(N)8...3 T T C T(N)7...5 300 u 1500 u

1 ml 1 ml
Concentration
5u/l
Note
MboII is blocked by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099). Greater than 15fold overdigestion with MboII may result in star activity. MboII produces DNA fragments that have a single-base 3extension which are more difficult to ligate than blunt-ended fragments. MboII may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
#ER0821 #ER0822

1XBuffer B at 37C.
Storage Buffer
MboII is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest MboII

After 5fold overdigestion with MboII, more than 80% of the DNA fragments can be ligated and more than 80% of these can be recut.
Methylation Effects
McrI MfeI MflI MjaIV
Fermentas enzymes FastDigest BsiEI (Bsh1285I) and Bsh1285I (BsiEI) Fermentas enzymes FastDigest MfeI (MunI) and MunI (MfeI) Fermentas enzymes FastDigest PsuI and PsuI (BstYI) Fermentas enzymes FastDigest Hpy8I and Hpy8I (MjaIV)
127
1
MlsI (MscI)
5...T 3...A
G GC C A...3 C CG G T...5 200 u 1000 u

1 ml 1 ml
Concentration
5 u/
Methylation Effects
#ER1211 #ER1212

1XBuffer R at 37C.

Storage Buffer
MlsI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest MscI (MlsI)
Note
MlsI is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with MlsI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
MluI
5...AC 3...T
G C G T...3 G C G CA...5 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with MluI, more than 95% of the DNA fragments can be ligated and recut.
#ER0561

1XBuffer R at 37C.
Methylation Effects
Storage Buffer
MluI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
#ER0562
5000 u
2x1 ml 1 ml

Also available as FastDigest MluI
MluNI MlyI
Fermentas enzymes FastDigest MscI (MlsI) and MlsI (MscI) Fermentas enzymes FastDigest MlyI (SchI) and SchI (MlyI)
MnlI
5...C 3...G
C T C(N)7...3 G A G(N)6...5 300 u 1500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER1071 #ER1072

1XBuffer G at 37C.
Note
MnlI produces DNA fragments that have a single-base 3extension which are more difficult to ligate than blunt-ended fragments. MnlI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Storage Buffer
MnlI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest MnlI

After 50fold overdigestion with MnlI, approximately 90% of the DNA fragments can be ligated and more than 90% of these can be recut.
www.fermentas.com
128
Mph1103I (NsiI)
T G C AT...3 3...TA C G T A ...5
5...A
Concentration
10u/l

After 50fold overdigestion with Mph1103I, more than 95% of the DNA fragments can be ligated and recut.
#ER0731
1000 u
1 ml 1 ml

1XBuffer R at 37C.
Storage Buffer
Mph1103I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 200 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
#ER0732
5000 u
2x1 ml 1 ml
Also available as FastDigest NsiI (Mph1103I)
MreI (Sse232I)
5...C 3...G
GC C G G C G...3 C G G C CG C...5 300 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with MreI, more than 95% of the DNA fragments can be ligated and recut.
#ER2021

1XBuffer G at 37C.
Storage Buffer
MreI is supplied in: 10 mM potassium phosphate (pH7.0 at 25C), 200 mM KCl, 0.1 mM EDTA, 0.01% TritonX-100, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest MreI
MroI MscI MseI

Fermentas enzymes FastDigest Kpn2I and Kpn2I (BspEI) Fermentas enzymes FastDigest MscI (MlsI) and MlsI (MscI) Fermentas enzymes FastDigest Tru1I and Tru1I (MseI); Fermentas enzyme FastDigest MseI (SaqAI) Fermentas enzymes FastDigest MslI (RseI) and RseI (MslI)
MslI
MspI (HpaII)
5...CC 3...G
G G...3 G CC...5 3000 u

2X1 ml
Concentration
10u/l

After 50fold overdigestion with MspI, more than 95% of the DNA fragments can be ligated and recut.
#ER0541 #ER0542

Methylation Effects
5x3000 u
2x1 ml
Storage Buffer
MspI is supplied in: 10 mM potassium phosphate (pH7.5 at 25C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Note
MspI is an isoschizomer of HpaII. When the external C in the sequence CCGG is methylated, MspI and HpaII cannot cleave. However, unlike HpaII, MspI can cleave the sequence when the internal C residue is methylated.
Also available as FastDigest MspI
129
1
MssI (PmeI)
5...G 3...C
T T TA A A C...3 A A AT T T G...5 250 u 1250 u

1 ml 1 ml
Concentration
5u/l
Methylation Effects
#ER1341 #ER1342

1XBuffer B at 37C.

Storage Buffer
MssI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest MssI

After 10fold overdigestion with MssI, more than 90% of the DNA fragments can be ligated and more than 90% of these can be recut.
MstI
Fermentas enzymes FastDigest FspI (NsbI) and NsbI (FspI)
MunI (MfeI)
5...CA 3...G
Activity in Five Buffer System, % B G O R Tango 2XTango 100 100 0-20 0-20 100 0-20
A T T G...3 T T A AC...5 300 u 1500 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with MunI, more than 95% of the DNA fragments can be ligated and recut.
#ER0751 #ER0752

1XBuffer G at 37C.
Storage Buffer
MunI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest MfeI (MunI)

Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 20-50 50-100 100 20-50* 100
* Star activity appears at a greater than 5fold overdigestion (5u x 1h).
MvaI (BstNI)
CW G G...3 3...G G WC C ...5
5...C
Concentration
10u/l 2000 u
1 ml 1 ml
Methylation Effects
#ER0551

1XBuffer R at 37C.
Note
Low salt, high glycerol (>5%) concentrations or a large excess of enzyme may result in star activity. Unlike its neoschizomer EcoRII, MvaI does not require multiple copies of recognition site for efficient cleavage.
Storage Buffer
MvaI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest MvaI

After 10fold overdigestion with MvaI, more than 90% of the DNA fragments can be ligated and more than 90% of these can be recut.
www.fermentas.com
130
Mva1269I (BsmI)
5...G
A A T G C N...3 3...C T T A CG N ...5 200 u 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with Mva1269I, more than 95% of the DNA fragments can be ligated and recut.
#ER0961 #ER0962

1XBuffer R at 37C.
Storage Buffer
Mva1269I is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Methylation Effects
Also available as FastDigest Mva1269I

MvnI MwoI NaeI NarI

Fermentas enzymes FastDigest Bsh1236I and Bsh1236I (BstUI) Fermentas enzymes FastDigest HpyF10VI and HpyF10VI (MwoI) Fermentas enzymes FastDigest NaeI (PdiI) and PdiI (NaeI) Fermentas enzymes FastDigest EheI and EheI (SfoI) (different cleavage position); Fermentas enzyme SspDI (KasI) (different cleavage position) Fermentas enzymes FastDigest NciI (BcnI) and BcnI (NciI)
NciI
NcoI
5...CC 3...G
A T G G...3 G T A CC...5 500 u 1000 u 2500 u

1 ml
Concentration
10u/l
Methylation Effects
#ER0571 #ER0575 #ER0572


Storage Buffer
NcoI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v)glycerol.
Also available as FastDigest NcoI
Note

After 50fold overdigestion with NcoI, more than 95% of the DNA fragments can be ligated and recut.
131
1
NdeI
5...C 3...G
AT A T G...3 T A TA C...5 500u 2500 u

1ml 1ml
Concentration
10u/l

After 50fold overdigestion with NdeI, more than 95% of the DNA fragments can be ligated and recut.
#ER0581 #ER0582

1XBuffer O at 37C.
Both supplied with: 10XBuffer O 10XBuffer Tango Both supplied with: 10XBuffer O 10XBuffer Tango
Storage Buffer
NdeI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
#ER0585
4000 u
2x1ml 1ml

Also available as FastDigest NdeI,
NdeII NgoMIV
Fermentas enzymes FastDigest Sau3AI (Bsp143I) and Bsp143I (Sau3AI) (different sensitivity to methylation); FastDigest DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest MboI and MboI Fermentas enzymes FastDigest NaeI (PdiI) and PdiI (NaeI) (different cleavage position)
NheI
5...GC 3...C
T A G C...3 G A T CG...5 500 u 1000 u 2500 u

1 ml
Concentration
10u/l

#ER0971 #ER0975 #ER0972

Storage Buffer
NheI is supplied in: 10 mM Tris-HCl (pH8.0 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity. NheI is inhibited by salt concentrations above 100 mM. Supercoiled plasmids may require up to 10fold more NheI for complete digestion than linear DNAs (e.g. 10 units are required to cleave 1g of pBR322 DNA).
Also available as FastDigest NheI

After 50fold overdigestion with NheI, more than 95% of DNA fragments can be ligated and recut.
Methylation Effects
NlaIII NlaIV
Fermentas enzymes FastDigest NlaIII (Hin1II) and Hin1II (NlaIII) Fermentas enzymes FastDigest NlaIV (BspLI) and BspLI (NlaIV)
www.fermentas.com
132
NmuCI (Tsp45I)
5...G 3...
T S A C ...3 C A S T G...5 200 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with NmuCI, more than 95% of the DNA fragments can be ligated and recut.
#ER1511

1XBuffer R at 37C.
Storage Buffer
NmuCI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest NmuCI
NotI
5...G 3...C
CG G C C G C...3 G C C G GC G...5 300 u 500 u 1500 u HC, 1500 u

1 ml 1 ml
Concentration
10u/l 50u/l, HC
Methylation Effects
#ER0591 #ER0595 #ER0592 #ER0593

1XBuffer O at 37C.

Minimum 5units of the enzyme are required for complete digestion of 1g of agarose-embedded Adenovirus-2 DNA in 16hours.
Storage Buffer
NotI is supplied in: 20 mM Tris-HCl (pH7.8 at 25C), 100 mMNaCl, 0.1 mM EDTA, 1 mM DTT, 0.02% TritonX-100, 0.2mg/mlBSA and 50% (v/v)glycerol.
All supplied with: 10XBuffer O 10XBuffer Tango
Note
Supercoiled plasmids may require up to 5fold more NotI for complete digestion than linear DNAs.
Also available as FastDigest NotI

After 50fold overdigestion with NotI, more than 95% of DNA fragments can be ligated and recut.
NruI
Fermentas enzymes FastDigest NruI (RruI) and Bsp68I (NruI)
NsbI (FspI)
5...T 3...A
G CG C A...3 C GC G T...5 400 u

1 ml
Concentration
10u/l

After 50fold overdigestion with NsbI, more than 80% of the DNA fragments can be ligated and more tha 95% of these can be recut.
#ER1221

Storage Buffer
NsbI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest FspI (NsbI)

NsiI NspI NspV
Fermentas enzymes FastDigest Nsil (Mph1103I) and Mph1103I (NsiI) Fermentas enzymes FastDigest NspI (XceI) and XceI (NspI) Fermentas enzymes FastDigest Bsp119I and Bsp119I (BstBI)
133
1
OliI (AleI)
5...C 3...G
A C N NN N G T G...3 T G N NN N C A C...5 200 u 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with OliI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
#ER1631 #ER1632

1XBuffer R at 37C.
Storage Buffer
OliI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest AleI (OliI)

PacI
5...T 3...A
T A A TT A A...3 A TT A A T T...5 250 u 1250 u

1 ml
Concentration
10u/l
Methylation Effects
#ER2201 #ER2202

1XBuffer PacI at 37C.

Minimum 5units of the enzyme are required for complete digestion of 1g of agarose-embedded pJET1 DNA with inserted PacI recognition sequence in 16hours.
Supplied with: 10XBuffer PacI
Storage Buffer
PacI is supplied in: 10 mM potassium phosphate (pH7.5 at 25C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% glycerol.
Also available as FastDigest PacI

After 50fold overdigestion with PacI, more than 95% of the DNA fragments can be ligated and recut.
PaeI (SphI)
5...G 3...C G
C A T GC...3 T A C G...5 500 u 2500 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with PaeI, more than 95% of the DNA fragments can be ligated and recut.
#ER0601 #ER0602

1XBuffer B at 37C.
Methylation Effects
Storage Buffer
PaeI is supplied in: 10 mM potassium-phosphate (pH7.0 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.15%TritonX-100, 0.5mg/ml BSA and 50% (v/v)glycerol.
Also available as FastDigest SphI (PaeI)

PaeR7I
Fermentas enzymes FastDigest XhoI and XhoI
www.fermentas.com
134
PagI (BspHI)
A T G A...3 3...A G T A C T ...5 #ER1281 #ER1282 400 u 2000 u
1 ml 5...TC
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 50-100 100 NR NR NR
Concentration
10u/l
Methylation Effects
Dam, EcoBI: may overlap cleavage impaired (p.176, 181). Dcm, CpG, EcoKI no effect.

1XBuffer O at 37C.
Both supplied with: 10XBuffer O
Storage Buffer
PagI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

Also available as FastDigest BspHI (PagI)
Note
Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity. PagI cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with PagI, more than 95% of the DNA fragments can be ligated and recut.
PasI
5...C 3...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR NR NR NR NR
CC W G G G...3 G G W CC C...5 200 u

1 ml
Concentration
10u/l

After 10fold overdigestion with PasI, more than 90% of the DNA fragments can be ligated and recut.
#ER1861

1XBuffer PasI at 55C.
Supplied with: 10XBuffer PasI
Methylation Effects
Storage Buffer
PasI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

Note
Incubation at 37C results in 30% activity. Greater than 10fold overdigestion with PasI results in star activity.
PauI (BssHII)
5...GC 3...C
G C G C...3 G C G CG...5 200 u 1000 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER1091 #ER1092

1XBuffer R at 37C.

Storage Buffer
PauI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest BssHII (PteI)
Note
Low salt, high glycerol (>5%) concentrations or a large excess of enzyme may result in star activity.

After 50fold overdigestion with PauI, more than 95% of the DNA fragments can be ligated and recut.
135
1
PciI
Fermentas enzyme PscI (PciI)
PdiI (NaeI)
5...G 3...C
C CG G C...3 G GC C G...5 200 u 1000 u

1 ml
Concentration
10u/l
Methylation Effects
#ER1521 #ER1522


Minimum 20 units of the enzyme are required for complete digestion of 1g of agaroseembedded pBR322 DNA in 16hours.
Storage Buffer
PdiI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 500 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA, 0.15%TritonX-100 and 50% (v/v)glycerol.
Also available as FastDigest NaeI (PdiI)
Note
For cleavage with PdiI at least two copies of its recognition sequence are required. Certain sites in pBR322 are difficult to cleave with PdiI, the same as with its prototype NaeI.

After 50fold overdigestion with PdiI, more than 90% of the DNA fragments can be ligated and recut.
PdmI (XmnI)
5...G 3...C
A A N NN N T T C...3 T T N NN N A A G...5 500 u 2500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with PdmI, more than 95% of the DNA fragments can be ligated and recut.
#ER1531 #ER1532

Methylation Effects
Storage Buffer
PdmI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 5 mM MgCl2, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest PdmI

PfeI (TfiI)
5...G A 3...C
W T C...3 T W AG...5 500 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with PfeI, more than 95% of the DNA fragments can be ligated and recut.
#ER1781

1XBuffer O at 37C.
Storage Buffer
PfeI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 250 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Dam, Dcm, EcoKI no effect. CpG: may overlap blocked (p.179). EcoBI: may overlap effect not determined.
Also available as FastDigest TfiI (PfeI)
www.fermentas.com
136
Pfl23II (BsiWI)
T A C G...3 3...G C A T GC ...5 #ER0851 300 u
1 ml Supplied with: 10XBuffer Tango 5...CG
Concentration
3u/l

After 10fold overdigestion with Pfl23II, more than 95% of the DNA fragments can be ligated and recut.

Methylation Effects
Storage Buffer
Pfl23II is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest BsiWI (Pfl23II)

PflFI PflMI
Fermentas enzymes FastDigest PsyI and PsyI (Tth111I) Fermentas enzymes FastDigest PflMI (Van91I) and Van91I (PflMI)
PfoI
5...TC 3...A
C N G G A...3 G G N C CT...5 200 u

1 ml
Concentration
10u/l
Methylation Effects
Dam: may overlap cleavage impaired (p.176). Dcm, CpG: may overlap blocked (p.177, 179). EcoKI, EcoBI no effect.
#ER1751

Storage Buffer
PfoI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

Also available as FastDigest PfoI
Note
PfoI cleavage is impaired by overlapping dam methylation and blocked by overlapping dcm methylation. To avoid dam or dcm methylation, use a dam, dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with PfoI, more than 95% of the DNA fragments can be ligated and recut.
PhoI PinAI PleI PmaCI PmeI PmlI
Fermentas enzymes FastDigest HaeIII (BsuRI) and BsuRI (HaeIII) Fermentas enzymes FastDigest AgeI (BshTI) and BshTI (AgeI) Fermentas enzymes FastDigest MlyI (SchI) and SchI (MlyI) (different cleavage position) Fermentas enzymes FastDigest PmlI (Eco72I) and Eco72I (PmlI) Fermentas enzymes FastDigest MssI and MssI (PmeI) Fermentas enzymes FastDigest PmlI (Eco72I) and Eco72I (PmlI)
137
1
PpiI
5... 7(N)G 3...12(N)C
A A C(N)5C T C(N)13...3 T T G(N)5G A G(N) 8 ...5 50 u

1 ml 1 ml
Concentration
2u/l
Methylation Effects
#ER1541

1XBuffer R at 30C.
Note
Incubation at 37C results in 60% activity. PpiI cleaves certain DNA sequences at random 7 or 8nt away on the top strand of the recognition sequence: 5...7-8(N) G A A C (N)5 C T C (N)13...3
3... 12(N) C T T G (N)5 G A G (N)8 ...5
Storage Buffer
PpiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

After 10fold overdigestion with PpiI, more than 90% of the DNA fragments can be ligated and recut.
The presence of SAM in a reaction mixture results in incomplete cleavage with PpiI. Greater than 10fold overdigestion with PpiI may result in star activity.
Ppu21I (BsaAI)
A CG T R...3 3...R T GC A Y...5
5...Y
Concentration
10u/l
Activity in Five Buffer System, % B G O R Tango 2XTango 50-100* 100* 20-50 NR NR NR
Methylation Effects
#ER1971
500 u
1 ml

1XBuffer Ppu21I at 30C.
Supplied with: 10XBuffer Ppu21I
Storage Buffer
Ppu21I is supplied in: 10 mM potassium phosphate (pH7.4 at 25C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.

Also available as FastDigest BsaAI (Ppu21I)
Note
Incubation at 37C results in less than 30% activity. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.

After 50fold overdigestion with Ppu21I, more than 90% of the DNA fragments can be ligated and recut.
PpuMI
Fermentas enzymes FastDigest PpuMI (Psp5II) and Psp5II (PpuMI)
PscI (PciI)
5...AC 3...T
A T G T...3 G T A CA...5 200 u 1000 u

1 ml
Concentration
10u/l
Methylation Effects
#ER1871 #ER1872


Storage Buffer
PscI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
PscI, like its isoschizomers BspLU11I and PciI, is inhibited by nonionic detergents TritonX-100 (>0.002%) and Nonidet P40 (>0.001%).

After 50fold overdigestion with PscI, more than 95% of the DNA fragments can be ligated and recut.
www.fermentas.com
138
1. CONVENTIONAL RESTRICTION ENZYMES PshAI PshBI PsiI

Fermentas enzymes FastDigest PshAI (BoxI) and BoxI (PshAI) Fermentas enzymes FastDigest AseI (VspI) and VspI (AseI) Fermentas enzymes FastDigest PsiI (AanI) and AanI (PsiI)
1
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 100 20-50 20-50 50-100 100
Psp5II (PpuMI)
5...R 3...Y
GG W C C Y...3 C C W GG R...5 500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER0761

1XBuffer G at 37C.
Storage Buffer
Psp5II is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.15%TritonX-100, 0.2mg/mlBSA and 50% (v/v)glycerol.

Also available as FastDigest PpuMI (Psp5II)
Note
Psp5II is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with Psp5II, more than 95% of the DNA fragments can be ligated and recut.
Psp1406I (AclI)
5...A 3...T
AC G T T...3 T G CA A...5 300 u 1500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with Psp1406I, more than 95% of the DNA fragments can be ligated and recut.
#ER0941 #ER0942

Storage Buffer
Psp1406I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest AclI (Psp1406I)

PspGI PspOMI

Fermentas enzymes EcoRII, FastDigest MvaI and MvaI (BstNI) (different cleavage position and different sensitivity to methylation) Fermentas enzymes FastDigest ApaI and ApaI (different cleavage position); FastDigest Bsp120I and Bsp120I (PspOMI)
139
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PstI
5...C 3...G A
T G C AG...3 C G T C...5 3000 u

2x1 ml 1 ml
Concentration
10u/l

#ER0611

1XBuffer O at 37C.
Supplied with: 10XBuffer O 10XBuffer Tango Supplied with: 10XBuffer O 10XBuffer Tango Supplied with: 10XBuffer O 10XBuffer Tango
Storage Buffer
PstI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 200 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.15%TritonX-100, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Conditions of high pH, low salt, high glycerol, 8% DMSO can cause star activity (Malyguine, E., et al., Gene, 8, 163-177, 1980). Surrounding sequences: the presence of adjacent runs of G-C base pairs confers significant resistance to cleavage (Armstrong, K. and Bauer, W.R., NAR, 10, 993-1007, 1982). 100% dUTP incorporation at the recognition site reduces PstI cleavage to 25% (Glenn, T.C., et al., Biotechniques, 17, 1086-1090, 1994). PstI will not cut AGCTGCAG when methylated by AluI methyltransferase.
#ER0615
10000 u
4x1 ml 1 ml
#ER0612
5x3000 u
2x1 ml 1 ml

After 50fold overdigestion with PstI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest PstI
Methylation Effects
Dam, Dcm,CpG, EcoKI, EcoBI no effect.
PsuI (BstYI)
5...R G 3...Y
A T C Y...3 C T A GR...5 500 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with PsuI, more than 95% of the DNA fragments can be ligated and recut.
#ER1551

1XBuffer B at 37C.
Methylation Effects
Storage Buffer
PsuI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest PsuI
Note
High glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
PsyI (Tth111I)
5...G 3...C
A C NN N G T C...3 T G N NN C A G...5 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with PsyI, more than 60% of the DNA fragments can be ligated and more than 95% of these can be recut.
#ER1331

1XBuffer B at 37C.
Storage Buffer
PsyI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest PsyI

www.fermentas.com
140
PvuI
5...C
G A TC G...3 3...G C T A G C ...5 300 u 1500 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with PvuI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
#ER0621 #ER0622

1XBuffer R at 37C.
Storage Buffer
PvuI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest PvuI

Activity in Five Buffer System, % B G O R Tango 2XTango 50-100* 100 20-50 50-100 20-50* 20-50*
PvuII
5...C
A GC T G...3 3...G T C G A C ...5 2500 u

1 ml 1 ml Supplied with: 10XBuffer G 10XBuffer Tango
Concentration
10u/l 50u/l, HC
Methylation Effects
#ER0631

1XBuffer G at 37C.

#ER0635 #ER0633
5000 u HC, 12500 u

2x1 ml 1 ml
Storage Buffer
PvuII is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Greater than 15fold overdigestion with PvuII may result in star activity.
Also available as FastDigest PvuII

After 10fold overdigestion with PvuII, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
RcaI
Fermentas enzymes FastDigest BspHI (PagI) and PagI (BspHI)
RsaI
5...G 3...C
TA C...3 AT G...5 1000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with RsaI, more than 95% of the DNA fragments can be ligated and recut.
#ER1121 #ER1122

Methylation Effects
5000 u
2x1 ml
Storage Buffer
RsaI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest RsaI
141
1
RseI (MslI)
5...C 3...G
A Y N NN N R T G...3 T R N NN N Y A C...5 200 u 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with RseI, more than 95% of the DNA fragments can be ligated and recut.
#ER2001 #ER2002

1XBuffer R at 37C.
Methylation Effects
Dam, Dcm, CpG no effect. EcoKI: may overlap blocked (p.181). EcoBI: may overlap effect not determined.
Storage Buffer
RseI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest MslI (RseI)

RsrII
Fermentas enzymes FastDigest RsrII (CpoI) and CpoI (RsrII)
SacI
5...G 3...C T
A G C TC...3 C G A G...5 1200 u 2000 u

1 ml 1 ml
Concentration
10u/l
Note
SacI is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC. AluI methyltransferase (AGmCT) can be used to block SacI. Supercoiled plasmids may require up to 5fold more SacI for complete digestion than linear DNA. SacI is inhibited by common clinical anticoagulants found in some preparation of anticoagulated peripheral blood and bone marrow. Levels of EDTA and ACD (citric acidsodium citrate-dextrose) in standard sample preparation have been shown to inhibit SacI. Three times the normal concentration for heparin is required to inhibit SacI. (Coad, J.E., et al., Inhibition of restriction endonucleases by common clinical anticoagulants, Anal. Biochem., 205, 368-369,1992).
#ER1131 #ER1135

1XBuffer SacI at 37C.
Both supplied with: 10XBuffer SacI 10XBuffer Tango Supplied with: 10XBuffer SacI 10XBuffer Tango
Storage Buffer
SacI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
#ER1132
5x1200 u
2x1 ml 1 ml

After 50fold overdigestion with SacI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest SacI
Methylation Effects

SacII
Fermentas enzyme Cfr42I (SacII)
www.fermentas.com
142
SalI
C G A C...3 3...C A G C TG ...5 #ER0641 #ER0645 1500 u 2000 u
1 ml 1 ml 5...GT
Concentration
10u/l
Methylation Effects

1XBuffer O at 37C.

Both supplied with: 10XBuffer O 10XBuffer Tango Supplied with: 10XBuffer O 10XBuffer Tango
Storage Buffer
SalI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C),100mMKCl, 0.1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.
#ER0642
5x1500 u
2x1 ml 1 ml
Note
Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity. Supercoiled forms of pBR322 and pUC require 10fold overdigestion with SalI to achieve complete digestion. Incubation at 25C results in 50-75% activity.

After 50fold overdigestion with SalI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest SalI
SanDI SapI
Fermentas enzyme FastDigest SanDI (KflI) Fermentas enzymes FastDigest SapI (LguI) and LguI (SapI)
SatI (Fnu4HI)
5...G 3...C
CN G C...3 G NC G...5 200 u 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with SatI, more than 60% of the DNA fragments can be ligated and more than 90% of these can be recut.
#ER1641 #ER1642

1XBuffer G at 37C.
Storage Buffer
SatI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest Fnu4HI (SatI)
Note
At least two copies of SatI recognition site are required for efficient cleavage.
SauI Sau96I Sau3AI

Fermentas enzymes FastDigest Bsu36I (Eco81I) and Eco81I (Bsu36I) Fermentas enzymes FastDigest Sau96I (Cfr13I) and Cfr13I (Sau96I) Fermentas enzymes FastDigest Sau3AI (Bsp143I) and Bsp143I (Sau3AI); FastDigest DpnI and DpnI (different cleavage position and different sensitivity to methylation); FastDigest MboI and MboI (different sensitivity to methylation) Fermentas enzymes FastDigest SbfI (SdaI) and SdaI (SbfI)
SbfI
143
1
ScaI
5...A 3...T
G TA C T...3 C AT G A...5 1000 u

1 ml
Concentration
10u/l
Methylation Effects
#ER0431 #ER0432

1XBuffer ScaI at 37C.
Supplied with: 10XBuffer ScaI Supplied with: 10XBuffer ScaI

5000 u
2x1 ml
Storage Buffer
ScaI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5mg/ml BSA and 50% (v/v)glycerol.
Also available as FastDigest ScaI
Note
Conditions of low salt, high enzyme concentration, glycerol concentration > 5% or pH> 8.0 may result in star activity. Supercoiled plasmids may require up to 20fold more ScaI for complete digestion than linear DNAs.

After 50fold overdigestion with ScaI, more than 80% of the DNA fragments can be ligated and more than 90% of these can be recut.
SchI (MlyI)
5...G 3...C
A G T C(N)5...3 T C A G(N)5...5 1000 u

1 ml
Concentration
10u/l
Methylation Effects
Dam, Dcm,CpG, EcoKI no effect. EcoBI: may overlap effect not determined.
#ER1371

Note
Greater than 10fold overdigestion with SchI may result in star activity. SchI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Storage Buffer
SchI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest MlyI (SchI)

After 10fold overdigestion with SchI, approximately 90% of DNA fragments can be ligated and more than 90% of these can be recut.
ScrFI
Fermentas enzymes FastDigest ScrFI (Bme1390I) and Bme1390I (ScrFI)
SdaI (SbfI)
5...C 3...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR NR 0-20 0-20 NR 20-50
C T G C AG G...3 GA C G T C C...5 300 u 1500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER1191 #ER1192

1XBuffer SdaI at 37C.

Both supplied with: 10XBuffer SdaI 10XBuffer Tango
Storage Buffer
SdaI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest SbfI (SdaI)
Note
Greater than 5fold overdigestion with SdaI may result in star activity.

After 5fold overdigestion with SdaI, more than 95% of the DNA fragments can be ligated and recut.
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144
SduI (Bsp1286I)
D G C HC...3 3...C H C G D G ...5
5...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR 50-100* 50-100 0-20 NR NR

Concentration
10u/l

After 50fold overdigestion with SduI, more than 95% of the DNA fragments can be ligated and recut.
#ER0651
500 u
1 ml

1XBuffer SduI at 37C.
Supplied with: 10XBuffer SduI
Methylation Effects
Storage Buffer
SduI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest Bsp1286I (SduI)
Note
SecI SexAI
Fermentas enzymes FastDigest BsaJI (BseDI) and BseDI (BsaJI) Fermentas enzyme FastDigest SexAI (CsiI)
SfaAI (AsiSI)
5...G 3...C
C G A TC G C...3 G CT A G C G...5 1000 u

1 ml
Concentration
10u/l
Methylation Effects
#ER2091


Minimum 5units of the enzyme are required for complete digestion of 1g of agarose-embedded pJET1 DNA with inserted SfaAI recognition site in 16hours.
Storage Buffer
SfaAI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest AsiSI (SfaAI)

After 50fold overdigestion with SfaAI, more than 90% of the DNA fragments can be ligated and more than 95% of these can be recut.
SfaNI SfcI SfeI
Fermentas enzymes FastDigest SfaNI (BmsI) and LweI (SfaNI) Fermentas enzymes FastDigest SfcI (BfmI) and BfmI (SfcI) Fermentas enzymes FastDigest SfcI (BfmI) and BfmI (SfcI)
145
1
SfiI
5...G 3...C
G C C N N N NN G G C C...3 C G G NN N N N C C G G...5 1000 u

1 ml 1 ml
Concentration
10u/l

Minimum 5units of the enzyme are required for complete digestion of 1g of agarose-embedded Adenovirus-2 DNA in 16hours.
#ER1821

1XBuffer G at 50C.
Storage Buffer
SfiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 300 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.15%TritonX-100, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Incubation at 37C results in 10% activity. SfiI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163 (#M0099). For cleavage with SfiI at least two copies of its recognition sequence are required. The two sites can be on either the same or different DNA molecules. (Wertzell, L.M. et al., J. Mol. Biol., 248, 581-595, 1995.) Therefore also an oligonucleotide harboring a SfiI recognition site can be supplemented.
Also available as FastDigest SfiI

After 50fold overdigestion with SfiI, more than 95% of the DNA fragments can be ligated and recut.
Methylation Effects
SfoI

Fermentas enzymes FastDigest EheI and EheI (SfoI); Fermentas enzyme FastDigest SspDI (KasI) (different cleavage position) Fermentas enzymes FastDigest Bsp119I and Bsp119I (BstBI) Fermentas enzymes FastDigest AsiSI (SfaAI) and SfaAI (AsiSI)
SfuI SgfI
SgeI
5... m5C 3... * SgeI cleaves DNA targets containing 5-methylcytosine on one or both DNA strands
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 0-20 0-20 NR NR NR
N N G (N)9 ...3 G N N C (N)13...5*
Concentration
3u/l

A minimum of 3 units of the enzyme is required for complete digestion of 1 g of agarose-embedded lambda DNA in 16hours.

1XBuffer SgeI at 37C.
#ER2211
250 u
1 ml
Supplied with: 10XBuffer SgeI
Storage Buffer
SgeI is supplied in: 10mMTris-HCl (pH7.4 at 25C), 100mMNaCl, 1mMDTT, 1mMEDTA and 50%glycerol.
Note
DNA methylated by Dcm or CpG methyltransferases will be a substrate for SgeI. Greater than 3fold overdigestion with SgeI may result in nonspecific cleavage. At least two copies of SgeI recognition site are required for an efficient cleavage. Amount of the enzyme required for complete digestion of methylated DNA depends on the number of SgeI recognition sites. DNA cleavage products generated by target site cleavage facilitate the nonspecific cleavage by SgeI. Therefore, optimization of enzyme amount is recommended for DNA cleavage. pBR322 DNA isolated from E.coli dcm+ strain (#SD0041) can be used as a DNA cleavage efficiency control. SgeI cleaves all six dcm-methylated targets on pBR322 DNA.

After a 2fold overdigestion with SgeI, more than 80% of the DNA fragments can be ligated and recut..
Methylation Effects
Dam, EcoKI, EcoBI no effect. Dcm: always cleaves DNA methylated by Dcm methyltransferase (p.177). CpG: cleaves targets overlapping with CpG methylated sequences (p.179).
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146
SgrDI
GT C G A C G...3 3...G C A G C T G C...5
5...C
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 0-20 0-20 100 NR 100
Concentration
5u/l
Methylation Effects
#ER2031
200 u
1 ml 1 ml

1XBuffer R at 37C.

Storage Buffer
SgrDI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%(v/v) glycerol.
Note
Low salt concentration or large excess of the enzyme may result in star activity.

After 30fold overdigestion with SgrDI, more than 80% of the DNA fragments can be ligated. More than 95% of these can be recut.
SgsI (AscI)
5...G 3...C
GC G C G C C...3 C G C G CG G...5 300 u 1500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with SgsI, more than 95% of the DNA fragments can be ligated and recut.
#ER1891 #ER1892

Methylation Effects
Storage Buffer
SgsI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest AscI (SgsI)

SinI
Fermentas enzymes FastDigest AvaII (Eco47I) and Eco47I (AvaII)
SmaI
5...C 3...G
C CG G G...3 G GC C C...5 1200 u 2000 u

1 ml
Concentration
10u/l 50u/l, HC
Methylation Effects
#ER0661 #ER0665 #ER0662 #ER0663


5x1200 u HC, 6000 u

2x1 ml
Storage Buffer
SmaI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Incubation at 37C results in 50% activity. SmaI has a half-life of 15min at 37C. Incubation at 25C results in 100% activity. SmaI needs K+ to work for activity.
Also available as FastDigest SmaI

After 50fold overdigestion with SmaI, more than 90% of the DNA fragments can be ligated and recut.
147
1
SmiI (SwaI)
5...A 3...T
T T TA A A T...3 A A AT T T A...5 1000 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER1241

1XBuffer O at 30C.

Minimum 10 units of the enzyme are required for complete digestion of 1g of agaroseembedded Adenovirus-2 DNA in 16hours.
Storage Buffer
SmiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest SwaI (SmiI)
Note

After 50fold overdigestion with SmiI, more than 80% of the DNA fragments can be ligated and more than 95% of these can be recut.
SmlI
Fermentas enzyme SmoI (SmlI)
SmoI (SmlI)
5...C T 3...G
Y R A G...3 A R Y TC...5 200 u

1 ml
Concentration
10u/l
Methylation Effects
Dam: may overlap cleavage impaired (p.176). Dcm, CpG, EcoKI no effect. EcoBI: may overlap effect not determined.
#ER1981

Storage Buffer
SmoI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

Note
Incubation at 37C results in 10% activity. SmoI cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam, dcm strain such as GM2163 (#M0099). Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.

After 50fold overdigestion with SmoI, more than 95% of the DNA fragments can be ligated and recut.
SmuI (FauI)
5...C 3...G
C C G C(N)4 ...3 G G C G(N)6...5 50 u

1 ml
Concentration
2u/l

After 10fold overdigestion with SmuI, more than 95% of the DNA fragments can be ligated and recut.
#ER1691

Methylation Effects
Storage Buffer
SmuI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Greater than 40fold overdigestion with SmuI may result in star activity.
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148
1. CONVENTIONAL RESTRICTION ENZYMES SnaI SnaBI SpeI SphI SplI Sse232I Sse8387I
Fermentas enzymes FastDigest BstZ17I (Bst1107I) and Bst1107I (BstZ17I) Fermentas enzymes FastDigest SnaBI (Eco105I) and Eco105I (SnaBI) Fermentas enzymes FastDigest SpeI (BcuI) and BcuI (SpeI) Fermentas enzymes FastDigest SphI (PaeI) and PaeI (SphI) Fermentas enzymes FastDigest BsiWI (Pfl23II) and Pfl23II (BsiWI) Fermentas enzymes FastDigest MreI and MreI (Sse232I) Fermentas enzymes FastDigest SbfI (SdaI) and SdaI (SbfI)
SsiI (AciI)
5...CC 3...G
Activity in Five Buffer System, % B G O R Tango 2XTango NR 20-50 100 50-100 NR 100
G C...3 G CG...5 200 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with SsiI, approximately 95% of the DNA fragments can be ligated. No more than 50% of these can be recut due to asymmetric recognition sequence of SsiI.
#ER1791

1XBuffer O at 37C.
Storage Buffer
SsiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigestAciI (SsiI)
Note
SspI
5...A 3...T
A TA T T...3 T AT A A...5 500 u 2500 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER0771 #ER0772

1XBuffer G at 37C.

Storage Buffer
SspI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest SspI
Note
Greater than 15fold overdigestion with SspI may result in star activity.

After 10fold overdigestion with SspI, more than 90% of the DNA fragments can be ligated and recut.
SspBI
Fermentas enzymes FastDigest Bsp1407I and Bsp1407I (BsrGI)
149
1
SspDI (KasI)
5...G G 3...C
Activity in Five Buffer System, % B G O R Tango 2XTango 20-50 0-20 NR 0-20 100 20-50
C G C C...3 C G C GG...5 250 u

1 ml
Concentration
10u/l
Methylation Effects
#ER2191


Storage Buffer
SspDI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

After a 50fold overdigestion with SspDI, more than 95% of the DNA fragments can be ligated and recut.
StuI StyI StyD4I SwaI
Fermentas enzymes FastDigest StuI (Eco147I) and Eco147I (StuI) Fermentas enzymes FastDigest StyI (Eco130I) and Eco130I (StyI) Fermentas enzymes FastDigest ScrFI (Bme1390I) and Bme1390I (ScrFI) (different cleavage position) Fermentas enzymes FastDigest SwaI (SmiI) and SmiI (SwaI)
TaaI (HpyCH4III)
5...A 3...T
C NG T...3 GN C A...5 200 u 1000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with TaaI, approximately 90% of the DNA fragments can be ligated and recut.
#ER1361 #ER1362

1XBuffer Tango at 65C. To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Methylation Effects
Also available as FastDigest TaaI
Storage Buffer
TaaI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Tail (MaeII*)
* Unlike MaeII, TaiI produces DNA fragments with a 4-base 3-extension 5...
Activity in Five Buffer System, % B G O R Tango 2XTango 50-100 50-100 20-50 100 100 50-100
Concentration
10u/l

After 50fold overdigestion with TaiI, more than 95% of DNA fragments can be ligated and recut.
3... T
A C G T...3 G C A ...5 400 u 2000 u

1 ml 1 ml

1XBuffer R at 65C. To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Methylation Effects
#ER1141 #ER1142
Storage Buffer
TaiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
FastDigest TaiI
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150
TaqI
G A...3 3...A G C T ...5 #ER0671 3000 u
2x1 ml 1 ml Supplied with: 10XBuffer TaqI 10XBuffer Tango Supplied with: 10XBuffer TaqI 10XBuffer Tango 5...TC
Concentration
10u/l,

After 50fold overdigestion with TaqI, more than 95% of DNA fragments can be ligated and recut.

1XBuffer TaqI at 65C. To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Methylation Effects
#ER0672
5x3000 u
4x1 ml 1 ml
Storage Buffer
TaqI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5mg/ml BSA and 50% (v/v)glycerol.
Note
TaqI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099). Incubation at 37C results in 10% activity. We recommend using TaqI, HC (#ER0673) at 37C.
Also available as FastDigest TaqI
TasI (Tsp509I)
5... A 3...
A T T ...3 T T A A...5 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with TasI, more than 95% of DNA fragments can be ligated and recut.
#ER1351

1XBuffer B at 65C. To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Supplied with: 10XBuffer B 10XBuffer Tango Supplied with: 10XBuffer B 10XBuffer Tango
Methylation Effects
#ER1352
5000 u
2x1 ml 1 ml
Storage Buffer
TasI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Also available as FastDigest Tsp509I (TasI)
TatI
T A C W...3 3...W C A T G W...5 #ER1291 100 u
1 ml Supplied with: 10XBuffer Tango 5...WG
Activity in Five Buffer System, % B G O R Tango 2XTango NR 50-100* 20-50 20-50 100* 0-20
Concentration
5u/l

After 2fold overdigestion with TatI, more than 95% of the DNA fragments can be ligated and recut.
Methylation Effects
Also available as FastDigest TatI
Storage Buffer
TatI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Incubation at 37C results in 20% activity. Greater than 5fold overdigestion with TatI may result in star activity.
151
1
TauI
5...G 3...CG
C S GC...3 S C G...5 50 u 250 u

1 ml 1 ml
Concentration
3u/l
Methylation Effects
#ER1651 #ER1652

1XBuffer B at 55C.
Note
Incubation at 37C results in 30% activity. TauI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Storage Buffer
TauI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest TauI

After 10fold overdigestion with TauI, more than 90% of DNA fragments can be ligated and recut.
TfiI TliI
Fermentas enzymes FastDigest TfiI (PfeI) and PfeI (TfiI) Fermentas enzymes FastDigest XhoI and XhoI
Tru1I (MseI)
5...TT 3...A
A A ...3 A TT...5 300 u 1500 u HC, 1500 u

1 ml 1 ml
Concentration
10u/l 50u/l, HC

After 50fold overdigestion with Tru1I, more than 90% of DNA fragments can be ligated and recut.
#ER0981 #ER0982 #ER0983

1XBuffer R at 65C. To ensure higher efficiency of digestion, perform the cleavage reaction under paraffin oil.
Methylation Effects
Also available as FastDigest Tru1I
Storage Buffer
Tru1I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol. Fermentas enzymes FastDigest Tru1I and Tru1I (MseI); Fermentas enzyme FastDigest MseI (SaqAI)
Note
Incubation at 37C results in 10% activity. We recommend using Tru1I, HC (#ER0983) at 37C.
Tru9I

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152
TscAI (TspRI)
5...
N N C A S T G N N...3 3... N N G T S A C N N ...5 1000 u

Concentration
10u/l
Methylation Effects
#ER2101

Note
Incubation at 37C results in less than 5% activity. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity. TscAI produces DNA fragments with a 9-base 3-extension. To avoid atypical DNA band patterns, use 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Also available as FastDigest TspRI (TscAI)
Storage Buffer
TscAI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 200 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.

After 50fold overdigestion with TscAI, more than 95% of the DNA fragments can be ligated and recut.
TsoI
5...T 3...A
Activity in Five Buffer System, % B+SAM G+SAM O+SAM R+SAM Tango+SAM 2XTango+SAM NR 100 50-100 0-20 50-100 20-50
A R C C A (N)11...3 T Y G G T (N) 9...5 50 u

1 ml 0.1 ml 1 ml
Concentration
3u/l

After 10fold overdigestion with TsoI, approximately 80% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme.
#ER1991

1XBuffer G at 55C. SAM 0.05mM.
Supplied with: 10XBuffer G 50XSAM 10XBuffer Tango
Storage Buffer
TsoI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol. TsoI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. Low salt, high glycerol (>5%) concentrations, pH>8.0 or a large excess of enzyme may result in star activity.
Methylation Effects
Dam, Dcm, CpG, EcoBI no effect. EcoKI: may overlap effect not determined. TsoI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Note
Incubation at 37 results in 10% activity. TsoI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.05mM S-adenosylmethionine gives a 2fold increase in TsoI activity. Still, complete cleavage of some substrates with TsoI is difficult to achieve.
Tsp4CI Tsp45I Tsp509I TspMI

Fermentas enzymes FastDigest TaaI and TaaI (HpyCH4III) Fermentas enzymes FastDigest NmuCI and NmuCI (Tsp45I) Fermentas enzymes FastDigest Tsp509I (TasI) and TasI (Tsp509I) Fermentas enzymes Cfr9I (XmaI); FastDigest SmaI and SmaI (different cleavage position) Fermentas enzymes FastDigest TspRI (TscAI) and TscAI (TspRI)
TspRI
153
1
TstI
5...
8
(N)C A C (N)6T C C (N)12 ...3

7
Concentration
5u/l

After 10fold overdigestion with TstI, more than 80% of DNA fragments can be ligated and recut.
3...13(N)G T G(N)6A G G (N)
...5
#ER1911
100 u
1 ml 1 ml

1XBuffer R at 37C.
Methylation Effects
Dam: may overlap blocked (p.176). Dcm, CpG, EcoKI no effect. EcoBI: may overlap effect not determined.
Storage Buffer
TstI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol. cleavage with TstI. Greater than 10fold overdigestion with TstI may result in star activity. TstI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical

Minimum 5units of the enzyme are required for complete digestion of 1g of agarose-embedded DNA in 16hours. DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Note
TstI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099). The presence of Sadenosylmethionine in a reaction mixture results in incomplete
Tth111I
Fermentas enzymes FastDigest PsyI and PsyI (Tth111I)
Van91I (PflMI)
5...C 3...G
C A N N N NN T G G...3 G T NN N N N A C C...5 400 u 2000 u

1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER0711 #ER0712

1XBuffer R at 37C.

Storage Buffer
Van91I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest PflMI (Van91I)
Note
Van91I is blocked by overlapping dcm methylation. To avoid dcm methylation, use a dam , dcm strain such as GM2163 (#M0099).

After 50fold overdigestion with Van91I, more than 90% of DNA fragments can be ligated and recut.
VpaK11BI
Fermentas enzymes FastDigest AvaII (Eco47I) and Eco47I (AvaII)
VspI (AseI)
5...A 3...T
Activity in Five Buffer System, % B G O R Tango 2XTango 0-20 50-100 100 20-50 100 100
TT A A T...3 A A TT A...5 1000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with VspI, more than 95% of DNA fragments can be ligated and recut.
#ER0911

1XBuffer O at 37C.
Methylation Effects
Storage Buffer
VspI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
#ER0912
5000 u
2x1 ml 1 ml

Also available as FastDigest AseI (VspI)
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154
XagI (EcoNI)
C T N NN N N A G G...3 3...G G A N N N N N T C C ...5
5...C
Concentration
10u/l

After 50fold overdigestion with XagI, approximately 80% of DNA fragments can be ligated and more than 95% of these fragments can be recut.
#ER1301
1000 u
1 ml 1 ml

1XBuffer R at 37C.
Storage Buffer
XagI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest EcoNI (XagI)

XapI (ApoI)
5...R A 3...Y
A T T Y...3 T T A AR...5 500 u

1 ml
Concentration
10u/l
Methylation Effects
#ER1381


Storage Buffer
XapI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest XapI
Note
Greater than 15fold overdigestion with XapI may result in star activity.

After 10fold overdigestion with XapI, more than 95% of the DNA fragments can be ligated and recut.
XbaI
5...T 3...A
C T A G A...3 G A T C T...5 1500 u

1 ml
Concentration
10u/l 50u/l, HC
Methylation Effects
#ER0681 #ER0685 #ER0682 #ER0683


3000 u 5x1500 u HC, 7500 u

2x1 ml
Storage Buffer
XbaI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
XbaI is blocked by overlapping dam methylation. To avoid dam methylation, use a dam , dcm strain such as GM2163 (#M0099).
Also available as FastDigest XbaI

After 50fold overdigestion with XbaI, more than 95% of DNA fragments can be ligated and recut.
155
1
XceI (NspI)
5...R 3...YG
C A T GY...3 T A C R...5 500 u 2500 u

1 ml
Concentration
10u/l

After 50fold overdigestion with XceI, more than 95% of the DNA fragments can be ligated and recut.
#ER1471 #ER1472

Methylation Effects
Storage Buffer
XceI is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 300 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest NspI (XceI)
XhoI
5...CT 3...G
C G A G...3 A G C TC...5 2000 u

1 ml 1 ml
Concentration
10u/l 50u/l, HC
Methylation Effects
#ER0691

1XBuffer R at 37C.

#ER0695 #ER0692 #ER0693
5000 u 5x2000 u HC, 10000 u

2x1 ml 1 ml
Storage Buffer
XhoI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Note
Supercoiled plasmids may require up to 5fold more XhoI for complete digestion than linear DNA.

After 50fold overdigestion with XhoI, more than 95% of the DNA fragments can be ligated and recut.
Also available as FastDigest XhoI
XhoII XmaI

Fermentas enzymes FastDigest PsuI and PsuI (BstYI) Fermentas enzymes Cfr9I (XmaI); FastDigest SmaI and SmaI (different cleavage position) Fermentas enzymes FastDigest EagI (Eco52I) and Eco52I (EagI) Fermentas enzymes Cfr9I (XmaI); FastDigest SmaI and SmaI (different cleavage position)
XmaIII XmaCI

XmaJI (AvrII)
5...CC 3...G
T A G G...3 G A T CC...5 200 u 1000 u

1 ml
Concentration
10u/l

After 50fold overdigestion with XmaJI, more than 95% of the DNA fragments can be ligated and recut.
#ER1561 #ER1562

Storage Buffer
XmaJI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Methylation Effects
Also available as FastDigest AvrII (XmaJI)

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156
XmiI (AccI)
5...G
TM K A C...3 3...C A K MT G ...5 400 u 2000 u

1 ml 1 ml
Concentration
10u/l

After 50fold overdigestion with XmiI, more than 95% of the DNA fragments can be ligated and recut.
#ER1481 #ER1482

1XBuffer B at 37C.
Methylation Effects
Storage Buffer
XmiI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA and 50% (v/v)glycerol.
Also available as FastDigest AccI (XmiI)

XmnI XspI ZraI

Nicking Enzymes
Fermentas enzymes FastDigest PdmI and PdmI (XmnI) Fermentas enzymes FastDigest BfaI (FspBI) and FspBI (BfaI) Fermentas enzymes FastDigest AatII and AatII (different cleavage position)
Nb.Bpu10I
5...C 3...G
C T N A G C...3 G A N TC G...5 1000 u

1 ml 1 ml
Concentration
5u/l
Applications
Production of single-stranded circular DNA from supercoiled double-stranded plasmids in vitro with subsequent use in DNA sequencing, site-specific mutagenesis, etc. Creation of nested deletions. Vector preparation for ligation independent cloning method. Preparations of covalently closed, doublestranded linear DNA molecules.
#ER1681

1XBuffer R: 10 mM Tris-HCl (pH8.5 at 37C), 10 mM MgCl2, 100 mM KCl, 0.1mg/ml BSA. Incubate at 37C.
Storage Buffer
Nb.Bpu10I is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA, 50%glycerol.
Note
Nb.Bpu10I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
Nicking and Cleavage

Incubation of 10 u of enzyme with 1g pUC19 DNA (lacking the recognition sequence of Bpu10I) for 1 h at 37C in 50l reaction buffer results in <10% conversion to circular form. Incubation of 1 u of enzyme with 1g pBR322 DNA for 1 h at 37C in 50l reaction buffer results in <5% conversion to linear form.
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1
Nt.Bpu10I
5...C 3...G
CT N A G C...3 G A N T C G...5 1000 u

1 ml 1 ml
Concentration
5u/l
Applications
#ER2011

1XBuffer R: 10 mM Tris-HCl (pH8.5 at 37C), 10 mM MgCl2, 100 mM KCl, 0.1mg/ml BSA. Incubate at 37C.
Storage Buffer
Nt.Bpu10I is supplied in: 10 mM Tris-HCl (pH7.5 at 25C), 200 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA, 50%glycerol.
Note
Nt.Bpu10I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.

Incubation of 20 u of enzyme with 1g pUC19 DNA (lacking the recognition sequence of Bpu10I) for 1 h at 37C in 50l reaction buffer results in <3% conversion to circular form. No detectable conversion of pBR322 DNA (1g) to linear form was observed after incubation with 20 u of Nt.Bpu10I for 1 h at 37C in 50l reaction buffer.
Nb.Mva1269I
5...G 3...C
A A T G C...3 T T A CG...5 1000 u

1 ml 1 ml
Concentration
10u/l
Applications
#ER2051

1XBuffer O: 50 mM Tris-HCl (pH7.5 at 37C), 10 mM MgCl2, 100 mM NaCl and 0.1mg/ml BSA. Incubate at 37C.
Storage Buffer
Nb.Mva1269I is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2mg/mlBSA, 50%glycerol.

Incubation of 10 u of enzyme with 1g pUC19 DNA (lacking the recognition sequence of Mva1269I) for 16 h at 37C in 50l reaction buffer results in <3% conversion to circular form. Incubation of 10 u of enzyme with 1g pBR322 DNA for 16 h at 37C in 50l reaction buffer results in <1% conversion to linear form.
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Homing Enzyme
I-SceI is a site-specific homing enzyme encoded by a mitochondrial intron of Saccharomyces cerevisiae (1, 2). Intron-encoded endonucleases are proteins that promote the first step in mobility of the intron at the DNA level. They recognize and cleave an intronless allele of their cognate gene to insert a copy of the intron by a double-strand-break repair mechanism that results in the recipient allele also becoming intron-plus (3-5). Homing endonucleases recognize long, 14-40 base pairs sequences and are, therefore, extremely rare-cutting enzymes. They allow the introduction of a single or several double-strand breaks into complex genomes. This capability makes these enzymes powerful tools in high-resolution physical mapping, genome organization analysis, gene cloning, site-directed recombination and double-strand-break repair studies in diverse biological systems (4, 6).
I-SceI
5...T A G G G 3...A T C C CT A T T
A T A AC A G G G T A A T...3 G T C C C A T T A...5 250 u

1 ml 1 ml 1 ml
Concentration
10u/l
Methylation Effects
#ER1771

1XBuffer Tango: 33 mM Tris-acetate (pH7.9 at 37C), 10 mM Mg-acetate, 66 mM K-acetate and 0.1mg/ml BSA. Incubate at 37C.

Minimum 20 units of the enzyme are required for complete digestion of 1g of agaroseembedded pUC-I-SceI DNA in 1hour (see the protocol below).
Supplied with: 10XBuffer Tango 10XBuffer Tango (without Mg-acetate) 100mM Mg-acetate
Storage Buffer
I-SceI is supplied in: 10 mM Tris-HCl (pH7.4 at 25C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2mg/mlBSA and 50%glycerol.
Note
Homing enzymes do not have stringently defined recognition sequences. They can tolerate minor sequence changes, which only partially affect the cleavage reaction. I-SceI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6XDNA Loading Dye & SDS Solution (#R1151) for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis. Assayed using pUC-I-SceI DNA.

After 50fold overdigestion with I-SceI, more than 95% of the DNA fragments can be ligated and recut.
Digestion of the Agarose-embedded DNA with I-SceI

1. Immerse an agarose plug in 50-100l of the 1XTango buffer without Mg-acetate* (supplied with the
enzyme). The volume of the buffer should be sufficient to completely cover the plug.
2. Add 20 u of the enzyme. 3. Incubate 2hours on ice. 4. Add 1/10 volume of the 100 mM Mg-acetate solution (supplied with the enzyme). 5. Incubate at 37C for 1hour.
References 1. Colleaux, L., et al., Recognition and cleavage site of the intron-encoded omega transposase, Proc. Natl. Acad.Sci. U.S.A.,85, 6022-6026, 1988. 2. Monteihet, C., et al., Purification and characterization of the in vitro activity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron, Nucleic Acids Res., 18, 1407-1413, 1990. 3. Dujon, B., Group I introns as mobile genetic elements: facts and mechanistic speculations review, Gene, 82, 91-114, 1989. 4. Belfort, M., Roberts R.J., Homing endonucleases: keeping the house in order, Nucleic Acids Res., 25, 3379-3388, 1997. 5. Chevalier, B.S., Stoddard, B.L., Homing endonucleases:structural and functional insight into the catalysis of intron/intein mobility, Nucleic Acids Res., 29, 3757-3774, 2001. 6. Jasin, M., Genetic manipulation of genomes with rarecutting endonucleases, Trends in Genetics, 12, 224-228, 1996.
Note
* Diffusion of the enzyme in the absence of Mg-acetate prior to digestion is necessary, because I-SceI is unstable in the presence of Mg2+ ions.
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Protocols and Recommendations

1.5. DNA digestion
We recommend digesting 0.2-1.5g DNA with a 2fold to 10fold excess of enzyme in a total volume of 20l . A typical restriction enzyme digestion protocol is below. 1. Add the following reaction components in the order indicated:
Water, nuclease-free (#R0581) 10Xrecommended buffer for restriction enzyme Substrate DNA Restriction enzyme Total volume 16-16.5l 2l 1l (~1g) 0.5-1l (5-10 u) 20l
1.7. Setting up double digestion

1.7.1. Double digestion with FastDigest enzymes
For protocols and recommendations for FastDigest restriction enzymes see p.70.
1.7.5. Digestion with enzymes having different temperature optimums

For double digestion with enzymes working at different temperatures perform sequential DNA cleavage. The optimal reaction temperature for each restriction enzyme is indicated both in the product description and the Certificate of Analysis. Information about the activity of mesophilic and thermophilic restriction enzymes at 37C is given in Table 1.7 on p.167.
1.7.2. DoubleDigest Engine

DoubleDigest engine (www.fermentas/doubledigest.com) is a web tool for finding information on buffer and reaction conditions for double digests. Simply select two restriction enzymes required for digestion, submit the query and follow the recommendations.
2. Mix gently and spin down briefly. 3. Incubate at the optimal reaction temperature for 1-16hours.
The digestion reaction may be scaled either up or down. Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately. See Tables 1.5 and 1.6 for restriction enzyme buffer composition and reaction conditions, respectively.
1.7.6. Sequential digestion

If a double digest is not possible due to buffer incompatibility or star activity, perform sequential digestions in the optimal buffer for each enzyme. 1. Digest the DNA with the first restriction enzyme in its optimal buffer. 2. Purify the digested DNA by spin column or chloroform extraction and ethanol precipitation. 3. Digest the DNA with the second restriction enzyme in its optimal buffer.
Note
1.7.3. Double digestion in the universal Tango Buffer

Use Table 1.8 (p.168) to set up the optimal conditions for your double digestion reactions in the universal Tango buffer. 1. Determine the concentration of Tango buffer recommended for each restriction enzyme. 2. If the recommended concentration of Tango buffer is the same for both enzymes, the digests can be performed at the same time. 3. If the two restriction enzymes require different concentrations of Tango buffer, perform the digestions sequentially. Set-up the first digestion reaction (including both enzymes) in 1XTango buffer, then add an additional aliquot of the 10XTango buffer (1/8 of initial reaction volume) to attain a final 2XTango buffer concentration to optimize conditions for the second enzyme.
If both 1Xand the 2Xconcentrations of Tango buffer are suitable for the double digest, use the 2Xconcentrated buffer to reduce probability of star activity.
1.6. Digestion of PCR products

The most convenient option for digestion of PCR-amplified DNA is the addition of a restriction enzyme directly to the reaction tube after completion of PCR. The majority of Fermentas restriction enzymes are active in Fermentas PCR buffers. However, digestion of PCR products in the amplification mixture is often inefficient. Therefore, PCR reaction mixture should not make more than 1/3 volume of digestion reaction mixture to avoid inhibitory effects. 1. Add the following reaction components in the order indicated:
PCR reaction mixture Water, nuclease-free (#R0581) Restriction enzyme Total volume 10l (~0.1-0.5g of DNA) 16-17l 1-2l (10-20 u) 30l
1.8. Stability during prolonged incubation

The stability of restriction enzymes in a reaction mixture depends on the nature of the enzyme, the buffer composition and the incubation temperature. If a restriction enzyme retains its activity in the reaction mixture for more than onehour, DNA can be digested with less enzyme in a prolonged incubation period. The exact quantities of enzymes sufficient for overnight digestion are listed in the Table 1.6 on p.163.
Note
1.9. Dilution of restriction enzymes

Dilution Buffer for Restriction Enzymes (#B19) is available from Fermentas for applications that require diluted enzymes. Enzymes diluted in this buffer retain 50-100% activity after storage for one month at -20C.
10Xrecommended buffer for restriction enzyme 2l *
1.7.4. Double digestion in Five Buffer System

Table 1.6 (p.163) presents the activities of Fermentas restriction enzymes in the Five Buffer System. 1. Determine which color-coded buffers are recommended for each enzyme. 2. If possible, use the buffer in which both enzymes have 100% activity. 3. If this is not possible, choose the buffer in which both enzymes maintain at least 20% of their activity. Increase the amount of the enzymes in your digest according to their activity in that buffer.
* Only 2l of 10Xreaction buffer is required for unpurified PCR product in a 30l reaction volume.
2. Mix gently and spin down briefly. 3. Incubate at the optimal reaction temperature for 1-16hours.
1.10. Partial digestion of DNA

Certain cloning experiments may require incomplete DNA cleavage. Such partial digestion of the DNA can be achieved by using the following conditions: suboptimal concentration of the restriction enzyme in the reaction mixture, short incubation time, incubation at a suboptimal temperature. For certain targets, partial cleavage of the desired DNA site is inefficient due to site preferences of restriction enzymes (see Site Preferences by Restriction Endonucleases on p.173).
Note
For cloning applications, purification of PCR products prior to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the yield of ligation. If the restriction enzyme requires special additives (e.g.,SAM), reduce the amount of water appropriately. If cleavage of the PCR product is inefficient purify the PCR product with the GeneJET PCR Purification Kit (#K0702) prior to digestion. After digestion, gel-purify the PCR product with the GeneJET Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513) to remove short DNA fragments which compete with the insert in a ligation reaction.
Note
For enzymes that are prone to relaxation of target specificity use a buffer in which they do not exhibit star activity.
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1.11. Digestion of agaroseembedded DNA
1. Embed the substrate DNA in 1% low melting temperature agarose, 1 g / 30 l. 2. Prepare ~30 l agarose plugs with a GelSyringe or similar agarose dispensing system. 3. Equilibrate the plug in 100 l of the appropriate 1Xrestriction enzyme buffer for 15 min. 4. Place the plug in 100 l of fresh 1Xbuffer containing the restriction enzyme (seeTable1.9 on p.170 for recommended quantities of enzymes). 5. Incubate at 30-37C for mesophilic enzymes or at 50-55C for thermophilic enzymes for 4-16hours.
1.12. Inactivation of restriction enzymes

Inactivation of restriction enzymes following a digestion reaction is often required for downstream applications. Thermal inactivation is a convenient method used to terminate enzyme activity. The majority of restriction enzymes can be heat-inactivated at 65C or 80C in 20 min. Information on susceptibility of Fermentas restriction enzymes to thermal inactivation is listed in the Table 1.6 on p.163), as well as in product descriptions and certificates of analysis. All known restriction enzymes with exception of BfiI, BmuI and BmrI require Mg2+ for DNA cleavage. Thus, addition of EDTA (20mM final concentration) to the reaction mixture is an alternative method that can be used to halt digestion. EDTA is generally not compatible with most of downstream applications, therefore purification of the digested DNA using GeneJET PCR Purification Kit (#K0702) or by chloroform extraction (see p.358) is recommended.
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1
Reaction Conditions
Reaction Buffers
Our Five Buffer System ensures the optimum reaction conditions for each restriction enzyme. This system consists of 10XB (blue), G (green), O (orange), R (red) and Tango (yellow) buffers (Table 1.6). All restriction enzymes are supplied in color-coded tubes to indicate the recommended reaction buffer. The recommended buffer and/or the universal Tango buffer, witch has been designed for double digestions of DNA, are supplied with each enzyme. Restriction enzyme buffers are also available separately. see Table 1.5 below for ordering information. For more information on double digestion see p.160 or visit the Fermentas DoubleDigest engine at www.fermentas.com/doubledigest. To ensure consistent enzyme performance, Fermentas restriction enzyme buffers contain BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations. Multiple freezethaw cycles of the buffers will not cause BSA precipitation. Fermentas restriction enzymes exhibit 100% of their certified activity in the recommended buffer. However, some enzymes require additives to achieve 100% activity. For example, AjuI, AlfI, BdaI, BplI, BseMII (BspCNI), Eco57I (AcuI), Eco57MI, FaqI (BsmFI), Hin4I, TsoI require S-adenosylmethionine, which is supplied with the enzyme, while AarI and BveI (BspMI) require an oligonucleotide (also supplied with the enzyme), and Esp3I requires DTT. The following enzymes require unique buffers for optimal digestion: AarI, AjiI (BmgBI), BamHI, BfuI (BciVI), Bpu10I, BseXI (BbvI), Bsp143I (Sau3AI), Cfr9I (XmaI), Cfr10I (BsrFI), Eam1105I (AhdI), Ecl136II (EcoICRI), Eco52I (EagI), EcoRI, KpnI, Lsp1109I (BbvI), PacI, PasI, Ppu21I (BsaAI), SacI, ScaI, SdaI (SbfI), SduI (Bsp1286I), SgeI and TaqI. The compositions of these unique buffers are listed in Table 1.5 as well as in of restriction enzymes Certificates of Analysis. All Fermentas restriction enzyme buffers should be stored at -20C.
Table 1.5. Reaction buffers for restriction enzymes.

10Xbuffer 1Xbuffer composition pH, Cat. Tris- TrisBis-Tris MgSodium Triton Gly BSA, at Quantity, MgCl , NaCl, KCl, K-acetate, EDTA, DTT, 2 # HCl, acetate, Propane-HCl, acetate, glutamate, X-100, cerol, mg/ 37C ml mM mM mM mM mM mM mM mM mM mM mM % % ml Five Buffer System 10XBuffer B 10XBuffer G 10XBuffer O 10XBuffer R 10XBuffer Tango Buffer Set for Restriction Enzymes 10XBuffer AarI, AjiI, Bpu10I, ScaI, PasI 10XBuffer BamHI, Lsp1109I, SgeI 10XBuffer BfuI 10XBuffer BseXI 10XBuffer Bsp143I 10XBuffer Cfr9I 10XBuffer Cfr10I 10XBuffer Eam1105I BB5 BG5 BO5 BR5 BY5 B30 5x1 5x1 5x1 5x1 5x1 1 ml of each buffer Unique buffers B27 B57 B59 B31 B13 B02 B04 B25 1 5x1 1 1 1 1 1 1 1 1 5x1 1 1 1 1 5x1 ml 10 10 10 10 50 10 37 3 5 150 100 100 1 1 50 10 10 10 10 50 33 5 5 5 10 3 10 10 15 150 100 100 0.02 0.02 100 100 10 50 2 100 10 66 200 0.02 0.02 10 10 5 100 100 15 100 0.02 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 6.5 8.0 7.9 7.5 7.9 7.2 8.0 7.5 6.5 8.5 7.5 7.5 7.0 7.2 8.0 7.4 10 10 50 10 33 10 10 10 10 50 100 100 10 66 0.1 0.1 0.1 0.1 0.1 7.5 7.5 7.5 8.5 7.9
10XBuffer Ecl136II, PacI, B26 SacI 10XBuffer Eco52I 10XBuffer EcoRI 10XBuffer KpnI 10XBuffer SdaI 10XBuffer SduI, Ppu21I 10XBuffer TaqI Dilution Buffer for Restriction Enzymes B22 B12 B29 B24 B23 B28 B19
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Recommended Reaction Conditions
Table 1.6. Reaction conditions for restriction enzymes. Enzyme AanI (PsiI) AarI AasI (DrdI) AatII Acc65I (Asp718I) AdeI (DraIII) AjiI (BmgBI) AjuI AlfI AloI (30C) AluI Alw21I (BsiHKAI) Alw26I (BsmAI) Alw44I (ApaLI) ApaI BamHI BauI (BssSI) BclI (55C) BcnI (NciI) BcuI (SpeI) BdaI (30C) BfiI (BmrI) BfmI (SfcI) BfuI (BciVI) BglI BglII Bme1390I (ScrFI) BoxI (PshAI) BpiI (BbsI) BplI Bpu10I Bpu1102I (BlpI) BseDI (BsaJI) (55C) BseGI (BtsCI) (55C) BseJI (BsaBI) (65C) BseLI (BslI) (55C) BseMI (BsrDI) (55C) BseMII (BspCNI) (55C) BseNI (BsrI) (65C) BseSI (Bme1580I) (55C) BseXI (BbvI) (65C) Bsh1236I (BstUI) Bsh1285I (BsiEI) Units for overnight incubation, u/g DNA 0.5 1.0 0.1 0.3 0.3 0.5 0.5 0.5 1.0 0.1 0.1 0.1 0.2 0.1 0.2 0.5 0.2 0.1 0.2 0.5 1.0 1.0 1.0 1.0 0.1 0.1 0.2 0.5 0.3 0.3 0.2 0.1 0.2 0.2 0.1 0.1 0.3 0.5 0.1 0.1 0.3 0.1 0.2 RecoThermal mmended inactivation buffer for in 20 min 100% activity 65 C 65 C 80 C 65 C 65 C NO 65 C 65 C 65 C 65 C 65 C 65 C 65 C 65 C 65 C 80 C (10 u) 65 C 80 C (10 u) 65 C NO 65 C 65 C 65 C 80 C 65 C NO 80 C 80 C 65 C 65 C 80 C 80 C 80 C 80 C NO NO 80 C 80 C 80 C 80 C (10 u) 80 C 65C 80 C Tango Unique (+oligo) B Tango O G Unique R (+SAM) R (+SAM) R Tango O Tango Tango B Unique Tango G Tango Tango G (+SAM) Tango Tango Unique O O O Tango G Tango (+SAM) Unique Tango Tango Tango O Tango R Tango (+SAM) B G Unique R G Enzyme activity in Fermentas buffers, % B (blue) 1X 50-100 NR 100 50-100 0-20 0-20 NR 0-20 (+SAM) 0-20 (+SAM) 0-20 50-100 0-20 50-100 50-100 100 20-50 * 0-20 20-50 20-50 50-100 NR 20-50 0-20 NR 0-20 0-20 20-50 0-20 20-50 0-20 (+SAM) 0-20 50-100 50-100 20-50 NR 20-50 0-20 50-100 (+SAM) 100 20-50 NR 0-20 20-50 G (green) O (orange) 1X 1X 50-100 NR 20-50 20-50 20-50 100 NR 50-100 (+SAM) 0-20 (+SAM) 0-20 0-20 20-50 100 100 20-50 100 50-100 100 50-100 50-100 100 (+SAM) 20-50 50-100 NR 50-100 20-50 50-100 0-20 100 20-50 (+SAM) 20-50 * 50-100 20-50 50-100 100 * 100 20-50 50-100 (+SAM) 20-50 100 NR 0-20 100 0-20 0-20 (+oligo) 0-20 0-20 100 20-50 20-50 * 20-50 (+SAM) 0-20 (+SAM) 0-20 0-20 100 0-20 0-20 0-20 20-50 0-20 20-50 50-100 0-20 0-20 (+SAM) 0-20 0-20 0-20 100 100 100 0-20 50-100 0-20 (+SAM) 50-100 * 20-50 0-20 20-50 100 50-100 0-20 50-100 (+SAM) 0-20 0-20 NR 50-100 20-50 R (red) 1X 0-20 0-20 (+oligo) 0-20 0-20 20-50 100 NR 100 (+SAM) 100 (+SAM) 100 0-20 50-100 0-20 50-100 0-20 50-100 * 50-100 20-50 50-100 20-50 20-50 (+SAM) 0-20 0-20 0-20 100 50-100 50-100 20-50 50-100 0-20 (+SAM) 100 * 20-50 0-20 20-50 NR 20-50 100 50-100 (+SAM) 0-20 20-50 NR 100 0-20 Tango (yellow) 1X 2X 100 NR 50-100 * 100 20-50 100 * NR 50-100 (+SAM) 0-20 (+SAM) 20-50 100 20-50 100 100 20-50 100 * 100 100 * 100 100 50-100* (+SAM) 100 100 NR 0-20 0-20 50-100 100 50-100 100 (+SAM) 50-100 * 100 100 100 NR 100 50-100 100 (+SAM) 50-100 50-100 NR 20-50 0-20 20-50 50-100 (+oligo) 0-20 20-50 50-100 20-50 20-50 * 50-100 (+SAM) 20-50 (+SAM) 100 20-50 50-100 100 50-100 0-20 50-100 50-100 100 50-100 0-20 50-100 (+SAM) 0-20 20-50 50-100 * 100 100 50-100 20-50 50-100 20-50 (+SAM) 100 * 20-50 50-100 20-50 100 * 50-100 50-100 50-100 (+SAM) 20-50 0-20 NR 50-100 20-50 Tango buffer for double digestion
1
1Xor 2X 2X (+oligo) 1X* 1Xor 2X 1Xor 2X 1X* or 2X 2X* 1Xor 2X (+SAM) 2X (+SAM) 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1X* or 2X 1Xor 2X 1X* or 2X 1Xor 2X 1X 1X* or 2X (+SAM) 1X 1Xor 2X 2X* 2X 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X (+SAM) 1X* or 2X* 1Xor 2X 1Xor 2X 1Xor 2X 2X* 1Xor 2X 1Xor 2X 1Xor 2X (+SAM) 1Xor 2X 1X NR 1Xor 2X 2X
163
Table 1.6.Reaction conditions for restriction enzymes.
1
Enzyme BshNI (BanI) BshTI (AgeI) Bsp68I (NruI) Bsp119I (BstBI) Bsp120I (PspOMI) Bsp143I (Sau3AI) Bsp1407I (BsrGI) BspLI (NlaIV) BspOI (BmtI) BspPI (AlwI) (55C) BspTI (AfIII) Bst1107I (BstZ17I) BstXI (55C) Bsu15I (ClaI) BsuRI (HaeIII) BveI (BspMI) CaiI (AlwNI) CfrI (EaeI) Cfr9I (XmaI) Cfr10I (BsrFI) Cfr13I (Sau96I) Cfr42I (SacII) CpoI (RsrII) CseI (HgaI) Csp6I (CviQI) DpnI DraI Eam1104I (EarI) Eam1105I (AhdI) Ecl136II (EcoICRI) Eco24I (BanII) Eco31I (BsaI) Eco32I (EcoRV) Eco47I (AvaII) Eco47III (AfeI) Eco52I (EagI) Eco57I (AcuI) Eco57MI Eco72I (PmlI) Eco81I (Bsu36I) Eco88I (AvaI) Eco91I (BstEII) Eco105I (SnaBI) Eco130I (StyI) Eco147I (StuI) EcoO109I (DraII) EcoRI
Units for overnight incubation, u/g DNA 0.3 0.1 0.2 0.1 0.1 0.1 0.1 0.3 0.2 0.5 0.1 0.1 0.1 0.1 0.1 0.2 0.2 1.0 0.2 0.1 0.3 0.1 0.5 0.5 0.1 0.1 0.1 0.5 0.1 0.2 0.2 0.3 0.1 0.3 0.1 0.2 1.0 1.0 0.5 0.1 0.2 0.1 0.5 0.2 0.1 0.2 0.2
Thermal inactivation in 20 min 65 C 80 C 65 C 80 C 80 C 65 C 65 C 65 C 80 C 80 C 65 C 80 C 80 C 65 C 80 C 65 C 65 C 65 C 65 C NO 65 C 65 C 65 C 80 C (10 u) 65 C 80 C 65 C 65 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C
Recommended buffer for 100% activity O O O Tango B Unique Tango Tango O Tango O O O Tango R O (+oligo) Tango Tango Unique Unique Tango B Tango R B Tango Tango Tango Unique Unique Tango G R R O Unique G (+SAM) B (+SAM) Tango Tango Tango O Tango O B Tango Unique
Enzyme activity in Fermentas buffers, % B (blue) 1X 0-20 0-20 0-20 20-50 100 20-50 0-20 50-100 0-20 20-50 0-20 20-50 20-50 20-50 20-50 0-20 (+oligo) 20-50 50-100 * 0-20 0-20 50-100 100 20-50 NR 100 100 50-100 50-100 20-50 50-100 50-100 50-100 0-20 0-20 0-20 0-20 100 (+SAM) 100 (+SAM) NR 50-100 100 20-50 100 * 0-20 100 50-100 0-20 G (green) O (orange) 1X 1X 20-50 20-50 20-50 0-20 20-50 20-50 20-50 50-100 0-20 20-50 0-20 50-100 100 20-50 20-50 20-50 (+oligo) 20-50 50-100 0-20 20-50 50-100 50-100 50-100 50-100 * 50-100 100 50-100 50-100 50-100 20-50 50-100 100 50-100 50-100 20-50 0-20 100 (+SAM) 50-100 (+SAM) NR 100 50-100 20-50 50-100 20-50 50-100 20-50 NR 100 100 100 0-20 0-20 0-20 0-20 0-20 100 0-20 100 100 100 20-50 50-100 100 (+oligo) 20-50 0-20 0-20 20-50 20-50 0-20 50-100 50-100 0-20 50-100 20-50 0-20 0-20 0-20 0-20 0-20 50-100 50-100 100 0-20 20-50 (+SAM) 0-20 (+SAM) 0-20 0-20 0-20 100 0-20 100 20-50 20-50 100 R (red) 1X 50-100 50-100 50-100 0-20 20-50 0-20 20-50 20-50 100 0-20 20-50 100 50-100 20-50 100 20-50 (+oligo) 50-100 0-20 0-20 50-100 * 20-50 0-20 20-50 100 0-20 50-100 20-50 0-20 0-20 0-20 20-50 0-20 100 100 100 20-50 20-50 (+SAM) 20-50 (+SAM) 0-20 0-20 0-20 50-100 0-20 50-100 20-50 20-50 100 * Tango (yellow) 1X 2X 0-20 20-50 20-50 100 50-100 50-100 100 100 0-20 100 0-20 20-50 50-100 100 50-100 50-100 (+oligo) 100 100 20-50 20-50 100 50-100 100 100 * 50-100 100 100 100 50-100 50-100 100 50-100 20-50 50-100 50-100 0-20 50-100 (+SAM) 50-100 (+SAM) 100 100 100 50-100 100 50-100 50-100 100 NR 100 20-50 50-100 100 0-20 20-50 50-100 20-50 20-50 0-20 50-100 100 100 20-50 100 100 (+oligo) 50-100 0-20 0-20 50-100 20-50 0-20 50-100 50-100 0-20 50-100 50-100 0-20 20-50 0-20 0-20 20-50 100 50-100 100 20-50 50-100 (+SAM) 0-20 (+SAM) 20-50 0-20 20-50 100 0-20 100 0-20 100 100
Tango buffer for double digestion 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1Xor 2X 1Xor 2X 2X 1X 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X (+oligo) 1Xor 2X 1X 1X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1X* or 2X 1X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1X 1X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 2X 1Xor 2X (+SAM) 1X (+SAM) 1Xor 2X 1X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1X 1Xor 2X 2X
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Table 1.6.Reaction conditions for restriction enzymes. Enzyme EcoRII EheI (SfoI) Esp3I (BsmBI) FaqI (BsmFI) FspAI FspBI (BfaI) GsuI (BpmI) (30C) HhaI Hin1I (BsaHI) Hin1II (NlaIII) Hin4I Hin6I (HinP1I) HincII (HindII) HindIII HinfI HpaII HphI Hpy8I (MjaIV) HpyF3I (DdeI) HpyF10VI (MwoI) KpnI Kpn2I (BspEI) (55C) KspAI (HpaI) LguI (SapI) Lsp1109I (BbvI) LweI (SfaNI) MauBI MbiI (BsrBI) MboI MboII MlsI (MscI) MluI MnlI Mph1103I (NsiI) MreI (Sse232I) MspI (HpaII) MssI (PmeI) MunI (MfeI) MvaI (BstNI) Mva1269I (BsmI) NcoI NdeI NheI NmuCI (Tsp45I) NotI NsbI (FspI) OliI (AleI) Units for overnight incubation, u/g DNA 0.1 1.0 0.2 1.0 0.2 0.3 1.0 0.1 0.1 0.3 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.2 0.5 0.5 0.5 0.2 0.1 0.2 0.1 1.0 0.5 0.1 0.5 0.3 0.5 0.3 0.5 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.2 Thermal inactivation in 20 min 80 C 65 C 65 C 80 C 65 C 65 C 65 C NO 65 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C 65 C 80 C 65 C 80 C 80 C 80 C 65 C 65 C 65 C 65 C 65 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C 80 C 80 C 65 C 65 C NO 65 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C Recommended buffer for 100% activity O Tango Tango (+DTT) Tango (+SAM) O Tango B Tango G G Tango (+SAM) Tango Tango R R Tango B Tango Tango Tango Unique Tango B Tango Unique Tango Tango Tango R B R R G R G Tango B G R R Tango O Tango R O Tango R Enzyme activity in Fermentas buffers, % B (blue) 1X 20-50 20-50 100 (+DTT) 20-50 (+SAM) 0-20 50-100 100 50-100 20-50 50-100 20-50 (+SAM) 50-100 50-100 0-20 0-20 50-100 100 50-100 20-50 0-20 20-50 50-100 100 20-50 0-20 0-20 0-20 20-50 50-100 100 0-20 0-20 50-100 0-20 20-50 50-100 100 100 20-50 0-20 20-50 0-20 100 0-20 0-20 20-50 0-20 G (green) O (orange) 1X 1X 50-100 50-100 20-50 (+DTT) 20-50 (+SAM) 0-20 20-50 50-100 50-100 100 100 20-50 (+SAM) 50-100 50-100 20-50 20-50 50-100 0-20 50-100 20-50 0-20 0-20 50-100 50-100 * 50-100 20-50 * 0-20 0-20 100 50-100 50-100 20-50 20-50 100 50-100 100 50-100 0-20 100 20-50 20-50 20-50 0-20 20-50 20-50 0-20 50-100 0-20 100 0-20 0-20 (+DTT) 0-20 (+SAM) 100 0-20 20-50 20-50 20-50 20-50 0-20 (+SAM) 20-50 20-50 0-20 50-100 0-20 0-20 0-20 20-50 0-20 0-20 0-20 20-50 20-50 50-100 * 0-20 0-20 20-50 50-100 20-50 0-20 50-100 20-50 20-50 0-20 0-20 0-20 0-20 50-100 50-100 20-50 100 0-20 50-100 100 0-20 0-20 R (red) 1X 50-100 0-20 0-20 (+DTT) 0-20 (+SAM) 50-100 0-20 20-50 20-50 20-50 50-100 0-20 (+SAM) 20-50 50-100 100 100 20-50 0-20 20-50 20-50 0-20 0-20 20-50 20-50 20-50 100 * 20-50 0-20 20-50 100 0-20 100 100 20-50 100 0-20 0-20 0-20 0-20 100 100 50-100 50-100 0-20 100 20-50 20-50 100 Tango (yellow) 1X 2X 20-50 100 100 (+DTT) 100 (+SAM) 0-20 100 100 100 20-50 50-100 100 (+SAM) 100 100 50-100 50-100 100 20-50 100 100 100 20-50 100 100 * 100 20-50 * 100 100 100 50-100 50-100 20-50 20-50 20-50 50-100 50-100 100 20-50 100 20-50 * 0-20 100 0-20 100 20-50 0-20 100 0-20 50-100 20-50 0-20 (+DTT) 20-50 (+SAM) 50-100 0-20 50-100 20-50 20-50 50-100 0-20 (+SAM) 50-100 50-100 50-100 50-100 20-50 0-20 50-100 50-100 50-100 0-20 50-100 50-100 20-50 20-50 * 20-50 0-20 20-50 100 20-50 50-100 50-100 20-50 50-100 0-20 50-100 0-20 0-20 100 50-100 100 50-100 0-20 50-100 20-50 20-50 50-100 Tango buffer for double digestion 1Xor 2X 1Xor 2X 1X (+DTT) 1Xor 2X (+SAM) 2X 1X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X (+SAM) 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1X* or 2X 1Xor 2X 1X* or 2X* 1Xor 2X 1X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1X 1X 1X* or 2X 2X 1Xor 2X 2X 1X 1Xor 2X 2X 1Xor 2X 2X
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Table 1.6.Reaction conditions for restriction enzymes.
1
Enzyme PacI PaeI (SphI) PagI (BspHI) PasI (55C) PauI (BssHII) PdiI (NaeI) PdmI (XmnI) PfeI (TfiI) Pfl23II (BsiWI) PfoI PpiI (30C) Ppu21I (BsaAI) (30C) PscI (PciI) Psp5II (PpuMI) Psp1406I (AclI) PstI PsuI (BstYI) PsyI (Tth111I) PvuI PvuII RsaI RseI (MslI) SacI SalI SatI (Fnu4HI) ScaI SchI (MlyI) SdaI (SbfI) SduI (Bsp1286I) SfaAI (AsiSI) SfiI (50C) SgeI SgrDI SgsI (AscI) SmaI (30C) SmiI (SwaI) (30C) SmoI (SmlI) (55C) SmuI (FauI) SsiI (AciI) SspI SspDI (KasI) TaaI (HpyCH4III) (65C) TaiI (MaeII) (65C) TaqI (65C) TasI (Tsp509I) (65C) TatI (65C) TauI (55C) Tru1I (MseI) (65C) TscAI (TspRI) (65C)
Units for overnight incubation, u/g DNA 0.3 0.1 0.2 0.2 0.2 0.5 0.5 0.1 0.3 0.1 0.1 0.5 0.2 0.2 0.5 0.2 0.5 0.1 0.2 0.2 0.2 0.5 0.2 0.1 0.1 0.5 0.2 0.3 0.3 0.2 0.2 NR 0.3 0.1 0.2 0.1 0.2 0.2 0.5 0.1 0.1 0.2 0.3 0.3 0.3 0.2 1.0 0.2 0.2
Thermal inactivation in 20 min 65 C 65 C 80 C 80 C 80 C 65C 65 C 65 C 65 C 65 C 65 C 65 C 65 C 80 C 65 C NO 80 C 80 C 80 C (10 u) NO 80 C 65 C 65 C 65 C 65 C 80 C (10 u) 65 C 80 C 65 C 80 C NO 65 C 65 C 65 C 65 C 65 C 80 C 65 C 65 C 65 C 80 C NO NO NO NO NO NO NO NO
Recommended buffer for 100% activity Unique B O Unique R Tango Tango O Tango Tango R Unique Tango G Tango O B B R G Tango R Unique O G Unique Tango Unique Unique Tango G Unique R Tango Tango O Tango Tango O G Tango Tango R Unique B Tango B R Tango
Enzyme activity in Fermentas buffers, % B (blue) 1X 20-50 100 0-20 NR 0-20 50-100 20-50 0-20 20-50 0-20 0-20 50-100 * 20-50 0-20 100 50-100 100 100 0-20 50-100 * 50-100 0-20 50-100 0-20 20-50 0-20 20-50 NR NR 50-100 50-100 0-20 0-20 0-20 50-100 0-20 50-100 50-100 NR 20-50 20-50 0-20 50-100 0-20 100 NR 100 50-100 50-100 G (green) O (orange) 1X 1X 20-50 50-100 50-100 NR 0-20 20-50 50-100 20-50 50-100 20-50 0-20 100 * 20-50 100 50-100 50-100 20-50 50-100 20-50 100 20-50 50-100 20-50 0-20 100 0-20 50-100 NR 50-100 * 0-20 100 0-20 0-20 0-20 0-20 0-20 20-50 50-100 20-50 100 0-20 0-20 50-100 20-50 50-100 50-100 * 50-100 50-100 50-100 0-20 0-20 100 NR 100 0-20 0-20 100 20-50 50-100 0-20 20-50 0-20 20-50 0-20 100 0-20 0-20 50-100 20-50 0-20 50-100 0-20 100 20-50 0-20 0-20 0-20 50-100 0-20 20-50 0-20 0-20 0-20 0-20 100 0-20 0-20 100 0-20 NR 0-20 20-50 20-50 20-50 20-50 0-20 20-50 20-50 R (red) 1X 0-20 0-20 NR NR 100 0-20 0-20 50-100 20-50 0-20 100 NR 0-20 20-50 20-50 100 0-20 0-20 100 50-100 0-20 100 0-20 20-50 20-50 0-20 0-20 0-20 0-20 0-20 0-20 NR 100 50-100 0-20 20-50 20-50 20-50 50-100 50-100 0-20 50-100 100 20-50 0-20 20-50 0-20 100 20-50 Tango (yellow) 1X 2X
Tango buffer for double digestion NR 1X NR NR 2X 1Xor 2X 1X 1Xor 2X 1X 1Xor 2X 1Xor 2X NR 1X 1Xor 2X 1X 1Xor 2X 1X 1X 1Xor 2X 1X* or 2X* 1X 1Xor 2X 1Xor 2X 2X 1Xor 2X NR 1X 2X NR 1X 1X NR 2X 1Xor 2X 1X 2X 1Xor 2X 1Xor 2X 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1X* 1X 1Xor 2X 1Xor 2X
0-20 50-100 NR NR 0-20 100 100 20-50 100 100 50-100 NR 100 50-100 100 50-100 50-100 50-100 50-100 20-50 * 100 20-50 50-100 0-20 50-100 0-20 100 NR NR 100 100 NR NR 100 100 0-20 100 100 NR 100 100 100 100 20-50 20-50 100* 20-50 50-100 100
0-20 0-20 NR NR 100 50-100 0-20 50-100 0-20 50-100 50-100 NR 0-20 100 0-20 50-100 0-20 0-20 100 20-50 * 0-20 100 20-50 50-100 20-50 0-20 0-20 20-50 NR 0-20 0-20 NR 100 50-100 0-20 20-50 20-50 20-50 100 20-50 20-50 100 50-100 20-50 0-20 0-20 0-20 100 20-50
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Table 1.6.Reaction conditions for restriction enzymes. Enzyme Units for overnight incubation, u/g DNA 1.0 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.2 0.1 0.3 0.3 0.3 0.5 Thermal inactivation in 20 min 80 C NO 65 C 65 C 65 C 80 C 65C 65 C 80 C 80 C 65C 80 C 65 C 80 C 65 C Recommended buffer for 100% activity G (+SAM) R R O R Tango Tango Tango R Tango B R R O Tango Enzyme activity in Fermentas buffers, % B (blue) 1X NR (+SAM) 0-20 0-20 0-20 0-20 50-100 50-100 50-100 0-20 20-50 100 0-20 0-20 0-20 50-100 G (green) O (orange) 1X 1X 100 (+SAM) 0-20 50-100 50-100 20-50 100 50-100 0-20 50-100 50-100 0-20 20-50 0-20 20-50 50-100 50-100 (+SAM) 0-20 50-100 100 50-100 0-20 20-50 0-20 50-100 50-100 0-20 20-50 100 100 50-100 R (red) 1X 0-20 (+SAM) 100 100 20-50 100 0-20 0-20 0-20 100 50-100 0-20 100 100 100 50-100 Tango (yellow) 1X 2X 50-100 (+SAM) 0-20 20-50 100 20-50 100 100 100 20-50 100 50-100 20-50 0-20 20-50 100 20-50 (+SAM) 100 50-100 100 50-100 20-50 50-100 0-20 100 50-100 20-50 50-100 50-100 50-100 50-100 Tango buffer for double digestion 1Xor 2X (+SAM) 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 1X 1Xor 2X 1Xor 2X 1Xor 2X 1Xor 2X 2X 1Xor 2X 1Xor 2X
TsoI (55C) TstI Van91I (PflMI) VspI (AseI) XagI (EcoNI) XapI (ApoI) XbaI XceI (NspI) XhoI XmaJI (AvrII) XmiI (AccI) Nicking enzymes Nb.Bpu10I Nt.Bpu10I Nb.Mva1269I Homing enzyme I-SceI
* star activity appears at a greater than 5fold overdigestion (5units x 1hour). NR buffer is not recommended, because of high star activity. NO no thermal inactivation. 80C (10 u) only small amounts of the restriction enzyme (up to 10 units) can be inactivated at 80C in 20 min.
Activity of Mesophilic and Thermophilic Enzymes at 37C

Table 1.7. Activity of mesophilic and thermophilic enzymes at 37C. Enzyme Optimal temperature AloI 30 BclI 55 BdaI 30 BseDI (BsaJI) 55 BseGI (BtsCI) 55 BseJI (BsaBI) 65 BseLI (BslI) 55 BseMI (BsrDI) 55 BseMII (BspCNI) 55 BseNI (BsrI) 65 BseSI (Bme1580I) 55 BseXI (BbvI) 65 BspPI (AlwI) 55 BstXI 55 GsuI (BpmI) 30 Kpn2I (BspEI) 55 PasI 55 Activity at 37C, % 20 50 30 10 25 <10 40 20 30 <10 20 10 30 50 70 50 30 Enzyme PpiI Ppu21I (BsaAI) SfiI SmaI* SmiI (SwaI) SmoI (SmlI) TaaI (HpyCH4III) TaiI (MaeII) TasI (Tsp509I) TaqI* TauI TatI Tru1I (MseI) * TscAI (TspRI) TsoI Optimal temperature 30 30 50 30 30 55 65 65 65 65 55 65 65 65 55 Activity at 37C, % 60 <30 10 50 70 10 10 <10 <10 10 30 20 10 <5 10
* high concentration enzyme preparations are available for incubation at non-optimal temperature.
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Double Digestion in Universal Tango Buffer

Table 1.8. Double digestion in universal Tango buffer. Buffers for double digestions Tango Recommended buffer 1X 2X Tango AanI (PsiI) +oligo AarI AarI +oligo NR B * AasI (DrdI) NR AatII Tango O Acc65I (Asp718I) G * AdeI (DraIII) AjiI * AjiI (BmgBI) NR +SAM +SAM AjuI R +SAM +SAM AlfI R +SAM NR AloI (30C) R AluI Tango O Alw21I (BsiHKAI) Tango Alw26I (BsmAI) Tango Alw44I (ApaLI) ApaI B NR BamHI BamHI * Tango BauI (BssSI) BclI (55C) G * Tango BcnI (NciI) Tango BcuI (SpeI) NR +SAM +SAM * BdaI (30C) G +SAM Tango BfiI (BmrI) NR Tango BfmI (SfcI) BfuI * BfuI (BciVI) NR BglI O NR BglII O NR O Bme1390I (ScrFI) Tango BoxI (PshAI) G BpiI (BbsI) +SAM BplI Tango+SAM NR Bpu10I Bpu10I * * Tango Bpu1102I (BlpI) Tango BseDI (BsaJI) (55C) Tango BseGI (BtsCI) (55C) O * NR BseJI (BsaBI) (65C) Tango BseLI (BslI) (55C) R BseMI (BsrDI) (55C) +SAM +SAM BseMII (BspCNI) (55C) Tango+SAM B BseNI (BsrI) (65C) NR BseSI (Bme1580I) (55C) G BseXI NR NR BseXI (BbvI) (65C) R Bsh1236I (BstUI) G Bsh1285I (BsiEI) NR O BshNI (BanI) NR O BshTI (AgeI) O Bsp68I (NruI) Tango Bsp119I (BstBI) B Bsp120I (PspOMI) NR Bsp143I Bsp143I (Sau3AI) Tango Bsp1407I (BsrGI) Tango BspLI (NlaIV) O BspOI (BmtI) NR Tango NR BspPI (AlwI) (55C) O BspTI (AflII) NR Fermentas enzyme Fermentas enzyme Bst1107I (BstZ17I) BstXI (55C) Bsu15I (ClaI) BsuRI (HaeIII) BveI (BspMI) CaiI (AlwNI) CfrI (EaeI) Cfr9I (XmaI) Cfr10I (BsrFI) Cfr13I (Sau96I) Cfr42I (SacII) CpoI (RsrII) CseI (HgaI) Csp6I (CviQI) DpnI DraI Eam1104I (EarI) Eam1105I (AhdI) Ecl136II (EcoICRI) Eco24I (BanII) Eco31I (BsaI) Eco32I (EcoRV) Eco47I (AvaII) Eco47III (AfeI) Eco52I (EagI) Eco57I (AcuI) Eco57MI Eco72I (PmII) Eco81I (Bsu36I) Eco88I (AvaI) Eco91I (BstEII) Eco105I (SnaBI) Eco130I (StyI) Eco147I (StuI) EcoO109I (DraII) EcoRI EcoRII EheI (SfoI) Esp3I (BsmBI) FaqI (BsmFI) FspAI FspBI (BfaI) GsuI (BpmI) (30C) HhaI Hin1I (BsaHI) Hin1II (NlaIII) Hin4I Hin6I (HinP1I) HincII (HindII) HindIII HinfI HpaII HphI Hpy8I (MjaIV) Buffers for double digestions Tango Recommended buffer 1X 2X O O Tango R +oligo +oligo O +oligo Tango Tango NR Cfr9I NR Cfr10I Tango B NR Tango R * B NR Tango Tango Tango NR Eam1105I Ecl136II NR Tango NR G R R O Eco52I NR +SAM +SAM G +SAM +SAM NR B +SAM Tango Tango NR Tango O Tango NR O B NR Tango EcoRI NR O Tango +DTT Tango+DTT NR +SAM +SAM Tango+SAM O NR Tango NR B Tango G G +SAM Tango+SAM NR Tango Tango R R Tango B NR Tango
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Table 1.8.Double digestion in universal Tango buffer. Fermentas enzyme HpyF3I (DdeI) HpyF10VI (MwoI) KpnI Kpn2I (BspEI) (55C) KspAI (HpaI) LguI (SapI) Lsp1109I (BbvI) LweI (SfaNI) MauBI MbiI (BsrBI) MboI MboII MlsI (MscI) MluI MnlI Mph1103I (NsiI) MreI (Sse232I) MspI (HpaII) MssI (PmeI) MunI (MfeI) MvaI (BstNI) Mva1269I (BsmI) NcoI NdeI NheI NmuCI (Tsp45I) NotI NsbI (FspI) OliI (AleI) PaeI (SphI) PacI PagI (BspHI) PasI (55C) PauI (BssHII) PdiI (NaeI) PdmI (XmnI) PfeI (TfiI) Pfl23II (BsiWI) PfoI PpiI (30C) Ppu21I (BsaAI) (30C) PscI (PciI) Psp5II (PpuMI) Psp1406I (AciI) PstI PsuI (BstYI) PsyI (Tth111I) PvuI PvuII RsaI RseI (MslI) SacI SalI SatI (Fnu4HI) ScaI Buffers for double digestions Tango Recommended buffer 1X 2X Tango Tango KpnI NR Tango B * Tango Lsp1109I * * Tango Tango NR Tango R B R R G R G NR Tango B NR G NR R * R NR Tango O NR Tango NR R O NR Tango R NR B NR PacI NR NR O NR NR PasI NR NR R NR Tango Tango NR O Tango NR Tango R Ppu21I NR NR Tango NR G Tango NR O B NR B NR R G * * Tango NR R SacI O NR G ScaI NR NR Fermentas enzyme SchI (MlyI) SdaI (SbfI) SduI (Bsp1286I) SfaAI (AsiSI) SfiI (50C) SgeI SgrDI SgsI (AscI) SmaI (30C) SmiI (SwaI) (30C) SmoI (SmoI) (55C) SmuI (FauI) SsiI (AciI) SspI SspDI (KasI) TaaI (HpyCH4III) (65C) TaiI (MaeII) (65C) TaqI (65C) TasI (Tsp509I) (65C) TauI (55C) TatI (65C) Tru1I (MseI) (65C) TscAI (TspRI) (65C) TsoI (55C) TstI Van91I (PflMI) VspI (AseI) XagI (EcoNI) XapI (ApoI) XbaI XceI (NspI) XhoI XmaJI (AvrII) XmiI (AccI) I-SceI Nb.Bpu10I Nt.Bpu10I Nb.Mva1269I Buffers for double digestions Tango Recommended buffer 1X 2X Tango NR SdaI NR SduI NR NR Tango NR G NR SgeI NR NR R NR Tango Tango NR O NR Tango Tango O NR G Tango Tango R TaqI B NR B NR Tango * NR R Tango +SAM +SAM G +SAM R NR R O R Tango Tango Tango NR R Tango B Tango R R NR O
* star activity appears at a greater than 5fold overdigestion (5units x 1hour). Optimal temperature for the enzymes listed is 37C unless otherwise indicated in parenthesis. NR buffer is not recommended since enzyme activity is less than 20% or star activity is too high.
Cleavage efficiency
20-50% 50-100%
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Digestion of Agarose-Embedded DNA

Table 1.9. Digestion of agarose-embedded DNA. Fermentas enzyme AanI (PsiI) AarI AasI (DrdI) AatII Acc65I (Asp718I) AdeI (DraIII) AjiI (BmgBI) AjuI AlfI Alw44I (ApaLI) ApaI BamHI BauI (BssSI) BcuI (SpeI) BfuI (BciVI) BglI BglII BoxI (PshAI) BpiI (BbsI) BplI Bpu1102I (BlpI) BseSI (Bme1580I) Bsh1285I (BsiEI) BshNI (BanI) BshTI (AgeI) Bsp68I (NruI) Bsp119I (BstBI) Bsp120I (PspOMI) Bsp1407I (BsrGI) BspOI (BmtI) BspTI (AflII) Bst1107I (BstZ17I) BstXI Bsu15I (ClaI) CaiI (AlwNI) CfrI (EaeI) Cfr9I (XmaI) Cfr10I (BsrFI) Cfr42I (SacII) DraI Eam1104I (EarI) Eam1105I (AhdI) Ecl136II (EcoICRI) Eco24I (BanII) Eco31I (BsaI) Eco32I (EcoRV) Eco47III (AfeI) Eco52I (EagI) Eco72I (PmlI) Eco81I (Bsu36I) Eco88I (AvaI) Eco91I (BstEII) Eco105I (SnaBI) Eco130I (StyI) Eco147I (StuI) Units of enzyme, needed for DNA cleavage in 16 h 5 5 5 5 5 10 5
5
Fermentas enzyme EcoO109I (DraII) EcoRI EheI (SfoI) Esp3I (BsmBI) FspBI (BfaI) Hin1I (BsaHI) HindIII KpnI Kpn2I (BspEI) (55C) KspAI (HpaI) LguI (SapI) MauBI MbiI (BsrBI) MlsI (MscI) MluI MssI (PmeI) MunI (MfeI) Mva1269I (BsmI) NcoI NdeI NheI NotI NsbI (FspI) OliI (AleI) PaeI (SphI) PacI PagI (BspHI) PasI (55C) PauI (BssHII) PdiI (NaeI) PdmI (XmnI) Pfl23II (BsiWI) PfoI Ppu21I (BsaAI) PscI (PciI) Psp5II (PpuMI) Psp1406I (AcII) PstI PsyI (Tth111I) PvuI PvuII RseI (MslI) SacI SalI ScaI SdaI (SbfI) SfaAI (AsiSI) SfiI (50C) SgeI SgrDI SgsI (AscI) SmaI (30C) SmiI (SwaI) (30C) SmoI (SmlI) (55C) SspI
5 5 5 5 5 10 5 5 5 10 5 10 5 5 5 5 20 5 5 5 10 20 5 5 5 5 5 5 5 5 20 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
Units of enzyme, needed for DNA cleavage in 16 h 5 5 20 5 10 5 5 5 5 5 5 5 5 20 5 5 5 5 5 5 5 5 5 5 5 5 5 10 10 20 10 5 10 5 5 5 10 5 5 5 5 5 5 5 20 5 5 5 3 5 5 5 10 5 5
Fermentas enzyme SspDI (KasI) TstI Van91I (PflMI) VspI (AseI) XagI (EcoNI) XapI (ApoI) XbaI XhoI XmaJI (AvrII) XmiI (AccI) I-SceI
Units of enzyme, needed for DNA cleavage in 16 h 5 5 5 5 5 5 5 5 10 5 20
Note
For digestion of agarose-embedded DNA protocol see1.11on p.161.
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Cleavage Efficiency Close to the Termini of PCR Fragments
Some restriction enzymes cleave DNA poorly when their recognition sites are located close to DNA termini. Table 1.10 lists the activities of Fermentas restriction enzymes when their target sites are located close to the end of a PCR product. Experiments were performed as follows: PCR primers were designed with 1-5 extra nucleotides at their 5-end adjacent to the recognition site for the restriction enzyme. The 5-end was labeled with [32P] by T4 Polynucleotide Kinase (#EK0031) and these labeled primers were used in the PCR reaction. PCR products were purified with the Silica Bead DNA Gel Extraction Kit (#K0513) and precipitated with ethanol. The PCR products (0.5g) were incubated with 10 units of restriction enzymes in the optimal buffer for 1hour at the recommended temperature. Reaction products were separated on 10% PAGE and the cleavage efficiency was determined using OptiQuant Image Analysis software.
Table 1.10. Cleavage efficiency close to the termini of PCR fragments.

Enzyme AanI AarI AasI AatII Acc65I AdeI AjiI AluI Alw21I Alw26I Alw44I ApaI BamHI BauI BcnI BclI BcuI BfiI BfmI BfuI BglI BglII Bme1390I BoxI BpiI Bpu10I Bpu1102I BseDI BseGI BseJI BseLI BseMI BseMII BseNI BseSI BseXI Bsh1236I Bsh1285I BshNI BshTI Bsp68I Bsp119I Bsp120I Bsp143I Bsp1407I BspLI BspOI BspPI BspTI Bst1107I BstXI Bsu15I BsuRI BveI CaiI CfrI Cfr9I Cfr10I Cfr13I Cfr42I CpoI CseI Csp6I DpnI DraI bp from the recognition site to fragment end 1 2 3 4 5 0 0-20 20-50 20-50 50-100 50-100 0 0-20 20-50 50-100 0-20 50-100 50-100 50-100 0-20 20-50 50-100 50-100 50-100 0 20-50 50-100 50-100 50-100 0-20 20-50 50-100 20-50 50-100 0 50-100 50-100 50-100 50-100 50-100 20-50 50-100 0 50-100 20-50 50-100 0 50-100 50-100 20-50 50-100 50-100 0 50-100 50-100 0 50-100 0 50-100 0-20 50-100 50-100 0 50-100 50-100 20-50 50-100 50-100 0-20 50-100 50-100 20-50 50-100 0 50-100 50-100 20-50 50-100 50-100 20-50 50-100 50-100 0 20-50 50-100 0 50-100 0 0-20 50-100 0-20 50-100 0 50-100 50-100 0-20 20-50 50-100 0-20 50-100 0 0-20 50-100 0 50-100 20-50 50-100 20-50 50-100 50-100 50-100 50-100 50-100 50-100 50-100 0 0-20 50-100 Enzyme Eam1104I Eam1105I Ecl136II Eco24I Eco31I Eco32I Eco47I Eco47III Eco52I Eco57I Eco57MI Eco72I Eco81I Eco88I Eco91I Eco105I Eco130I Eco147I EcoO109I EcoRI EcoRII EheI Esp3I FaqI FspAI FspBI GsuI HhaI Hin1I Hin1II Hin6I HincII HindIII HinfI HpaII HphI Hpy8I HpyF3I HpyF10VI KpnI Kpn2I KspAI LguI Lsp1109I LweI MauBI MbiI MboI MboII MlsI MluI MnlI Mph1103I MreI MspI MssI MunI MvaI Mva1269I NcoI NdeI* NheI NmuCI NotI NsbI bp from the recognition site to fragment end 1 2 3 4 5 0 50-100 0 50-100 50-100 50-100 20-50 50-100 20-50 50-100 50-100 0 0-20 50-100 0-20 50-100 50-100 50-100 0-20 50-100 50-100 50-100 20-50 50-100 20-50 50-100 0 50-100 0 50-100 50-100 50-100 0 0-20 20-50 50-100 20-50 50-100 50-100 0-20 50-100 0-20 20-50 50-100 20-50 50-100 50-100 50-100 50-100 0 0-20 50-100 0 0-20 50-100 50-100 0 0-20 50-100 50-100 0-20 50-100 0 50-100 0-20 50-100 20-50 50-100 50-100 50-100 0 50-100 20-50 50-100 50-100 0-20 50-100 20-50 50-100 0 0-20 20-50 0 0-20 50-100 50-100 50-100 0-20 50-100 20-50 50-100 0 50-100 20-50 50-100 0 20-50 50-100 0-20 50-100 20-50 50-100 20-50 50-100 0 20-50 50-100 0 50-100 0 50-100 0-20 20-50 50-100 0 20-50 50-100 20-50 50-100 20-50 50-100 0 0-20 50-100 Enzyme OliI PacI PaeI PagI PasI PauI PdiI PdmI PfeI Pfl23II PfoI Ppu21I PscI Psp5II Psp1406I PstI PsuI PsyI PvuI PvuII RsaI RseI SacI SalI SatI ScaI SchI SdaI SduI SfaAI SfiI SgrDI SgsI SmaI SmiI SmoI SmuI SsiI SspI SspDI TaaI TaiI TaqI TasI TatI TauI Tru1I TscAI TsoI Van91I VspI XagI XapI XbaI XceI XhoI XmaJI XmiI I-SceI bp from the recognition site to fragment end 1 2 3 4 5 0-20 20-50 50-100 20-50 50-100 0 0-20 20-50 50-100 20-50 50-100 50-100 0 50-100 0-20 50-100 50-100 50-100 0-20 20-50 50-100 0 20-50 50-100 50-100 0 50-100 0 50-100 0-20 50-100 0-20 50-100 0-20 50-100 0 50-100 20-50 50-100 50-100 50-100 0 0-20 20-50 50-100 50-100 20-50 50-100 0 50-100 0-20 50-100 50-100 0-20 50-100 50-100 0 0-20 20-50 50-100 0-20 20-50 50-100 50-100 50-100 0-20 50-100 0 50-100 50-100 50-100 0-20 50-100 20-50 50-100 20-50 50-100 50-100 0 20-50 50-100 0 20-50 50-100 0-20 20-50 50-100 20-50 50-100 0 0-20 50-100 0 50-100 20-50 50-100 0 50-100 50-100 20-50 50-100 20-50 50-100 20-50 50-100 0 50-100 0-20 50-100 50-100 50-100 50-100
* incubation was performed for 16hours.
Cleavage efficiency
0% 0-20% 20-50% 50-100%
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Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site
Close vicinity of restriction targets within multiple cloning sites (MSC) of vectors may have negative effect on the efficiency of double digestions within MSC. For efficient double digestion within MSC the first reaction should be performed with a restriction enzyme that cleaves DNA inefficiently when enzymes target is located close to the end of DNA. The second digestion should be performed with a restriction enzyme which tolerates a close proximity to the DNA end. Data presented below were generated using pUC19 plasmid DNA. The plasmid was cleaved with a primary restriction enzyme (the first cut) and end-labeled with [32P] by T4 Polynucleotide Kinase (#EK0031). The DNA was then digested for onehour with a second restriction enzyme (the second cut). Reaction products were separated by PAGE, and the amount of the label present on the DNA was determined by autoradiography. A decrease in radioactivity reflects cleavage efficiency of the second restriction enzyme.
pUC19 multiple cloning site

Eco24I Cfr9I HincII PstII XapI Ecl136II Acc65I Eco88I SalI BveI PaeI 396 EcoRI SacI KpnI SmaI BamHI XbaI XmiI SdaI HindIII 452 5 - C CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGG C - 3 3 - G GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG CAG ATC TCA GCT GGA CGT CCG TAC GTT CGA ACC G - 5
Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site.
Enzyme pair First cut Acc65I BamHI Acc65I Ecl136II Acc65I Eco24I Acc65I EcoRI Acc65I SacI Acc65I XapI BamHI Cfr9I BamHI Eco88I BamHI HincII BamHI KpnI BamHI SalI BamHI XbaI Cfr9I Ecl136I Cfr9I Eco24I Cfr9I SacI Cfr9I XbaI Ecl136II Eco88I Ecl136II EcoRI Ecl136II XapI Eco24I Eco88I Eco24I EcoRI Eco24I KpnI Eco24I XapI Eco88I SacI Eco88I XbaI Acc65I BamHI Acc65I Ecl136II Acc65I Eco24I Acc65I EcoRI Acc65I SacI Acc65I XapI BamHI Cfr9I BamHI Eco88I BamHI HincII BamHI KpnI BamHI SalI BamHI XbaI Cfr9I Ecl136II Cfr9I Eco24I Cfr9I SacI Cfr9I XbaI Ecl136II Eco88I Ecl136II EcoRI Ecl136II XapI Eco24I Eco88I Eco24I EcoRI Eco24I KpnI Eco24I XapI Eco88I SacI Eco88I XbaI Second cut BamHI Acc65I Ecl136II Acc65I Eco24I Acc65I EcoRI Acc65I SacI Acc65I XapI Acc65I Cfr9I BamHI Eco88I BamHI HincII BamHI KpnI BamHI SalI BamHI XbaI BamHI Ecl136II Cfr9I Eco24I Cfr9I SacI Cfr9I XbaI Cfr9I Eco88I Ecl136II EcoRI Ecl136II XapI Ecl136II Eco88I Eco24I EcoRI Eco24I KpnI Eco24I XapI Eco24I SacI Eco88I XbaI Eco88I Efficiency of the 2nd cut, % 100 100 100 50-100 100 0 100 100 50-100 0-20 50-100 50-100 50-100 50-100 100 50-100 50-100 100 100 100 50-100 100 20-50 100 50-100 100 50-100 100 50-100 50-100 50-100 50-100 100 50-100 100 100 50-100 50-100 100 50-100 100 100 50-100 0 50-100 20-50 100 50-100 100 100 bp from 1st cut* 4 4 1 3 1 1 7 7 1 1 7 7 0 0 0 0 7 9 4 4 7 7 1 1 5 7 5 5 5 5 6 6 7 5 3 1 3 1 5 5 1 1 1 1 1 1 5 5 6 6 Enzyme pair First cut EcoRI Cfr9I EcoRI Eco88I EcoRI KpnI EcoRI SacI HincII PaeI HincII PstI HincII SdaI HincII XbaI HindIII PaeI HindIII PstI HindIII SdaI KpnI Ecl136II KpnI SacI KpnI XapI PaeI PstI PaeI SalI PaeI SdaI PstI BamHI PstI SalI PstI XbaI SacI SmaI SacI XapI SalI SdaI SalI XbaI SdaI XbaI EcoRI Cfr9I EcoRI Eco88I EcoRI KpnI EcoRI SacI HincII PaeI HincII PstI HincII SdaI HincII XbaI HindIII PaeI HindIII PstI HindIII SdaI KpnI Ecl136II KpnI SacI KpnI XapI PaeI PstI PaeI SalI PaeI SdaI PstI BamHI PstI SalI PstI XbaI SacI SmaI SacI XapI SalI SdaI SalI XbaI SdaI XbaI Second cut Cfr9I EcoRI Eco88I EcoRI KpnI EcoRI SacI EcoRI PaeI HincII PstI HincII SdaI HincII XbaI HincII PaeI HindIII PstI HindIII SdaI HindIII Ecl136II KpnI SacI KpnI XapI KpnI PstI PaeI SalI PaeI SdaI PaeI BamHI PstI SalI PstI XbaI PstI SmaI SacI XapI SacI SdaI SalI XbaI SalI XbaI SdaI Efficiency of the 2nd cut, % 50-100 100 50-100 100 100 100 50-100 20-50 100 100 100 20-50 0-20 20-50 50-100 50-100 50-100 0 100 100 50-100 50-100 50-100 100 50-100 100 100 50-100 0 20-50 50-100 100 0 20-50 50-100 50-100 0 50-100 100 50-100 100 50-100 0-20 50-100 0 20-50 50-100 0-20 100 50-100 bp from 1st cut* 11 11 11 11 7 7 1 1 9 7 3 1 2 1 3 1 1 1 7 7 6 7 1 3 1 1 7 7 1 1 7 7 0 1 13 13 1 1 7 7 5 7 1 1 0 1 1 1 7 6 Enzyme pair First cut SmaI Acc65I SmaI BamHI SmaI Ecl136II SmaI Eco24I SmaI KpnI SmaI XbaI SmaI Acc65I SmaI BamHI SmaI Ecl136II SmaI Eco24I SmaI KpnI SmaI XbaI Second cut Acc65I SmaI BamHI SmaI Ecl136II SmaI Eco24I SmaI KpnI SmaI XbaI SmaI Efficiency of the 2nd cut, % 0-20 0 50-100 0-20 50-100 100 50-100 100 0 0 50-100 100 bp from 1st cut* 1 -1 2 0 7 7 7 5 1 -1 8 6
* only double-stranded portion of DNA is included, not the overhangs. simultaneous double digestion may be performed with these enzyme pairs. this enzyme should be used for the first cut this enzyme should be used for the second cut not recommended For reaction conditions see www.fermentas.com/doubledigest
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Common Properties
Classification of Restriction Enzymes
Restriction enzymes recognize specific nucleotide sequences and cleave DNA molecules either within or outside their recognition site. Digestion results in the generation of DNA with sticky (with 5- or 3-overhang) or blunt ends. The phenomenon of host specificity was first observed by Luria and Human in the early 1950s (1). Nearly a decade later, Arber and Dussoix predicted its molecular basis (2). They proposed that host specificity is based on a two-enzyme system: a restriction enzyme, which recognizes specific DNA sequences and cleaves foreign DNA upon its entrance into the bacterial cell, and a modification enzyme (methyltransferase), which protects the host DNA from degradation by its own restriction enzyme. Both restriction and modification enzymes recognize the same nucleotide sequence and together they form a restrictionmodification (R-M) system. Restriction-modification systems are widespread among bacteria and have also been isolated from phage, Archaea and eukaryotic algae viruses. R-M systems have been classified into four types (I, II, III and IV), depending on the complexity of their structure, cofactor requirements, mode of action and sequence cleaved (3). The best characterized and the most frequently used are type II restriction endonucleases. These enzymes recognize specific 4-8 bp DNA sequences and cleave a DNA sequence either within the recognition site, or at a specified position up to 20 base pairs outside the site. Often there is more than one enzyme that recognizes a particular nucleotide sequence. According to restriction enzyme nomenclature, an enzyme with a unique specificity which has been discovered first is called the prototype. Subsequently discovered enzymes with the same specificity are termed isoschizomers. An isoschizomer may differ from the prototype enzyme in site preferences, reaction conditions, as well as in sensitivity to methylation and exhibition of star activity. Restriction enzymes that recognize the same nucleotide sequence, but cleave DNA at different positions are called neoschizomers.
Site Preferences by Restriction Endonucleases

In 1975, Thomas and Davis discovered that EcoRI cleaves its five recognition sites on DNA at rates that differ by an order of magnitude (4). Similar examples have been documented for other restriction enzymes. Factors such as flanking sequences and the number of cleavage sites appear to influence cleavage efficiency (5). There are numerous restriction enzymes (EcoRII, NaeI, NarI, Ksp632I, BspMI, Eco57I, etc.), which never completely digest certain unmethylated target DNAs, even when using an excess of enzyme or prolonged incubation (6-9). Most of these enzymes are members of the expanding group of type II restriction enzymes which require simultaneous interaction with two copies of the target site for effective cleavage (10). These enzymes cleave DNA molecules with one recognition site very slowly. In the case of type IIE enzymes (EcoRII, NaeI), one of the target sequences serves as an allosteric effector for effective cleavage of the other recognition site (6, 11-13). Type IIF enzymes, (e.g., SfiI, Cfr10I, NgoMIV, BspMI) and majority of type IIB restriction enzymes (AlfI, AloI, BalI, BcgI, BsaXI, CspCI, FalI, PpiI, PsrI) cleave both recognition sequences in a concerted reaction (14-18). Type IIS enzymes, such as FokI, BpmI, BsgI and MboII, also interact with two copies of their recognition sequence before cleaving DNA by different mechanisms (19). Cleavage of resistant sites can be significantly enhanced by the addition of cleavable DNA, an oligodeoxyribonucleotide containing the recognition site, or spermidine (7, 9, 17, 20, 21). Different restriction enzymes recognizing the same nucleotide sequence (isoschizomers or neoschizomers) often do not effectively cleave the same resistant recognition site (e. g., Fermentas enzymes BveI, Cfr42I, Eam1104I and PdiI and their prototypes BspMI, SacII, Ksp632I and NaeI). However, some isoschizomers or neoschizomers cleave resistant sites at the same rate as normal sites. For example, EheI cleaves target DNA more efficiently than its prototype NarI. Thus, one recognition site of NarI on DNA and two sites on pBR322 are not cleaved to completion, even after incubation with 50 units of the enzyme for 16hours. Unlike NarI, Fermentas neoschizomer EheI cleaves DNA and pBR322 DNA completely under standard conditions.
References 1. Luria, S.E., Human, M.L., A nonhereditary, host-induced variation of bacterial viruses, J. Bacteriol., 64, 557-569, 1952. 2. Arber, W., Dussoix, D., Host specifi city of DNA producted by Escherichia Coli: I. Host controlled modifi cation of bacteriophage lambda, J. Mol. Biol., 5, 18-36, 1962. 3. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003. 4. Thomas M., Davis R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91, 315-328, 1975. 5. Sapienza P.J., et al., Thermodynamic and Kinetic Basis for the relaxed DNA Sequence Specificity of Promiscuous Mutant EcoRI endonuclease, J. Mol. Biol., 348, 307-324, 2005. 6. Kruger, D.H., et al., EcoRII can be activated to cleave refractory DNA recognition sites, Nucleic Acids Res., 16, 3997-4008, 1988. 7. Oller, A. R., et al., Ability of DNA and spermidine to affect the activity of restriction endonucleases from several bacterial species, Biochemistry, 30, 2543-2549, 1991. 8. Bolton, B.J., et al., Ksp632I: a novel class IIS restriction endonuclease from Kluyvera species 632 with the asymmetric hexanucleotide recognition sequence: 5-CTCTTCN-3 3-GAGAAGNNNN-5, Gene, 66, 31-43, 1988. 9. Reuter, M., et al., Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases, Anal. Biochem., 209, 232-237, 1993. 10. Halford, S.E., Hopping, jumping and looping by restriction endonucleases, Biochem. Soc. Trans, 29, 363-373, 2001. 11. Gabbara, S., Bhagwat, A.S., Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites, J.Biol.Chem., 267, 18623-18630, 1992. 12. Yang, C.C., Topal, M.D., Nonidentical DNA-binding sites of endonuclease NaeI recognize different families of sequences flanking the recognition site, Biochemistry, 31, 9657-9664, 1992. 13. Huai, Q., et al., Crystal structure of NaeI an evolutionary bridge between DNA endonuclease and topoisomerase, EMBO J., 19, 3110-3118, 2000. 14. Wentzell, L.M., et al., The SfiI restriction endonuclease makes a four-strand DNA break at two copies of its recognition sequence, J. Mol. Biol., 248, 581-595, 1995. 15. Siksnys, V., et al., The Cfr10I restriction enzyme is functional as a tetramer, J. Mol. Biol., 291, 1105-1118, 1999. 16. Deibert, M., et al., Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA, Nat. Struct. Biol., 7, 792-799, 2000. 17. Gormley, N.A., et al., The type IIs restriction endonuclease BspMI is a tetramer that acts concertedly at two copies of an asymmetric DNA sequence, J. Biol. Chem., 277, 40344041, 2002. 18. Marshall, J.J., et al., Restriction Endonucleases that Bridge and lexcise two recognition sites from DNA, J. Mol. Biol., 367, 419-431, 2007. 19. Bath, A.J., et al., Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA, J. Biol. Chem., 277, 4024-4033, 2002. 20. Conrad, M., Topal, M.D., DNA and spermidine provide a switch mechanism to regulate the activity of restriction enzyme NaeI, Proc. Natl.Acad Sci. USA, 86, 9707-9711, 1989. 21. Grigaite, R.J., et al., AarI, a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5-CACCTGC(N)4/8-3, Nucleic Acids Res., 30, e123, 2002.
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Star Activity
Restriction enzymes recognize specific nucleotide sequences within DNA molecules. However, their recognition site specificity can be reduced in vitro (1). Under certain conditions, enzymes are able to recognize and cleave nucleotide sequences which differ from the canonical site. At low ionic strength, for example, BamHI (with the recognition sequence GGATCC) is able to cleave the following sequences: NGATCC, GPuATCC and GGNTCC (2, 3). This phenomenon is called relaxed or star activity (4, 5). For most restriction enzyme applications, star activity is not desirable. Reports (4, 6-10) on star activity have suggested that this phenomenon is the result of: prolonged incubation (over digestion), high enzyme concentration in the reaction mixture (over digestion), high glycerol concentration in the reaction mixture, presence of organic solvents, such as ethanol, dimethyl sulfoxide (DMSO) or dimethyl formamide (DMFA), in the reaction mixture, low ionic strength of the reaction buffer, suboptimal pHvalues of the reaction buffer, substitution of Mg2+ for other divalent cations, such as Mn2+ or Co2+. In some cases, the termini generated by DNA cleavage with a restriction enzyme at the canonical site have been shown to stimulate enzyme star activity (11). Both star activity and incomplete DNA digestion result in atypical electrophoresis patterns that can be distinguished by careful examination of gel images (see Fig.1.4). Any tendency of a restriction enzyme to exhibit star activity is indicated both in the product description and in the Certificate of Analysis.
References 1. Nasri, M., Thomas, D., Relaxation of recognition sequence of specific endonuclease HindIII, Nucleic Acids Res., 14, 811-821, 1986. 2. George, J., Chirikjian, J.G., Sequence-specific endonuclease BamHI: Relaxation of sequence recognition, Proc. Natl. Acad. Sci. USA, 79, 2432-2436, 1982. 3. Kolesnikov, V.A., et al., Relaxed specificity of endonuclease BamHI as determined by identification of recognition sites in SV40 and pBR322 DNAs, FEBS Letters, 132, 101-103, 1981. 4. Polisky, B., et al., Specificity of substrate recognition by the EcoRI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 72, 3310-3314, 1975. 5. Woodbury, C.P., et al., DNA site recognition and reduced specificity of the EcoRI endonuclease, J. Biol. Chem., 255, 11534-11546, 1980. 6. George, J., et al., Sequence-specific endonuclease BamHI, J. Biol. Chem., 255, 6521-6524, 1980. 7. Malyguine, E., et al., Alteration of the specificity of restriction endonucleases in the presence of organic solvents, Gene, 8, 163-177, 1980. 8. Hsu, M., Berg, P., Altering the specificity of restriction endonuclease: effect of replacing Mg2+ with Mn2+, Biochemistry, 17, 131-138, 1978. 9. Mayer, H., Optimization of the EcoRI* activity of EcoRI endonuclease, FEBS Letters, 90, 341-344, 1978. 10. Nasri, M., Thomas, D., Alteration of the specificity of PvuII restriction endonuclease, Nucleic Acids Res., 15, 76777687, 1987. 11. Bitinaite, J., Schildkraut, I., Self generated DNA termini relax the specificity of SgrAI restriction endonuclease, Proc. Natl. Acad. Sci. USA, 99, 1164-1169, 2002.
How to distinguish between star activity and incomplete digestion?

Note
Star activity results in additional DNA bands below the expected bands and no additional bands above the largest expected fragment. These additional bands become more intense, while the expected bands become less intense, when either the incubation time or the amount of enzyme is increased. Incomplete DNA digestion results in additional bands above the expected DNA bands on the gel. Additional bands disappear when the incubation time or amount of enzyme is increased. No additional bands below the smallest expected fragment are observed. Certain restriction enzymes remain associated with the substrate DNA after cleavage and cause a change in the mobility of the digestion products during electrophoresis. The resulting atypical pattern is not related to star activity. In these cases, use a loading dye with SDS (6XDNA Loading Dye & SDS Solution, #R1151), heat the sample for 10min at 65C and chill on ice prior to loading on the gel (see p.433). This effect is characteristic of the following FastDigest enzymes: FastDigest BspMI (BveI), FastDigest HgaI (CseI), FastDigest AcuI (Eco57I), FastDigest FokI, FastDigest MboII, FastDigest TauI, FastDigest TspRI (TscAI) and Conventional restriction enzymes: AarI, AloI, BdaI, BseXI (BbvI), BveI (BspMI), CseI (HgaI), Eco57I (AcuI), Eco57MI, EcoRII, FaqI (BsmFI), GsuI (BpmI), Lsp1109I (BbvI), LweI (SfaNI), MboII, MnlI, SchI (MlyI), TauI, TscAI (TspRI), TsoI, TstI.
Complete digestion Incomplete cleavage Star activity
1 2 3 4 5 6 Figure 1.4. Enzyme complete digestion and star activity. 1 Lambda DNA. 2 Lambda DNA incubated 1hour with 0.15 u of EcoRI (incomplete cleavage). 3 Lambda DNA incubated 1hour with 0.4 u of EcoRI (incomplete cleavage). 4 Lambda DNA incubated 1hour with 1 u of EcoRI (complete digestion). 5 Lambda DNA incubated 16hours with 40 u of EcoRI (star activity). 6 Lambda DNA incubated 16hours with 70 u of EcoRI (star activity).
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Digestion of Methylated DNA
DNA methylation is the process of transferring a methyl group from a donor molecule to either a cytosine or an adenine by DNA methyltransferases. Such methylation is the most common and abundant DNA modification process in living organisms. Three types of methylated bases are predominantly found in DNA: 5-methylcytosine (m5C), N4-methylcytosine (m4C), N6-methyladenine (m6A). Other modified bases, such as 5-hydroxymethylcytosine (hm5C) and 5-hydroxymethyluracil (hm5U), have also been described. The organism-specific pattern of methylation depends on the methyltransferases specificity. In prokaryotes, DNA cleavage by a cognate restriction enzyme is prevented by the methylation of DNA by a sequence-specific methyltransferase which is an integral component of every restriction-modification (R-M) system (4, 5). The majority of E.coli strains used for propagation of plasmid DNA contain two site-specific DNA methyltransferases Dam and Dcm (6, 4). The methylase encoded by the dam gene methylates the N6-position of an adenine residue within the GATC sequence (8, 9). The methylase encoded by the dcm gene methylates the C5-position of the internal cytosine residue within the CCWGG sequence (7, 10). In addition to Dam and Dcm methylases, laboratory strains of E.coli K12 and B may contain EcoKI or EcoBI enzymes, respectively, encoded by a type I R-M system. These methyltransferases modify adenine residues within their respective recognition sequences: AAC(N)6GTGC for EcoKI and TGA(N)8TGCT for EcoBI (3, 4).
DNA from higher eukaryotic organisms possesses modified 5-methylcytosine residues within CpG or CpNpG contexts (5, 11-13). These tissue-specific methylation patterns are heritable. They participate in regulation of gene expression and cellular differentiation. In cases where a restriction enzyme target site overlaps a methylation site, the following digestion results are possible: no effect partial inhibition complete block The ability to cleave methylated DNA is an intrinsic and unpredictable property of each restriction enzyme. Therefore, isoschizomers and neoschizomers which recognize the same DNA sequences can differ in their sensitivity to DNA methylation (Table 1.12). For instance, MboI (recognition sequence GATC) does not cleave DNA methylated by Dam methylase (14), while the isoschizomer Bsp143I is insensitive to Dam methylation. EcoRII does not cleave DNA at the CCWGG site if it is methylated by Dcm, while its neoisoschizomer MvaI will cleave this methylated site (15). Thus, to achieve effective DNA digestion, it is necessary to take into account both the type of DNA methylation and the sensitivity of the restriction enzyme to that type of methylation. All restriction enzymes produced by Fermentas have been examined for their sensitivity to Dam, Dcm, CpG, EcoKI and EcoBI methylation of substrate DNA. Detailed methylation sensitivity information for our restriction enzymes is listed in the tables on pp.176-181, as well as in the product descriptions and Certificates of Analysis.
Table 1.12. Fermentas isoschizomers and neoschizomers with differing sensitivities to target methylation. Conventional Recognition and restriction enzyme Sensitivity to methylation cleavage sites couple Overlapping Dcm or CpG methylation may influence DNA cleavage Acc65I (Asp718I) GGTACC KpnI Not influenced by Dcm or CpG methylation GGTACC ApaI Overlapping Dcm or CpG methylation may influence DNA cleavage GGGCCC Blocked by overlapping Dcm or CpG methylation Bsp120I (PspOMI) GGGCCC GATC Not influenced by Dam, blocked by CpG methylation Bsp143I (Sau3AI) MboI GATC Blocked by Dam methylated DNA DpnI Cleaves only Dam methylated DNA GATC Not influenced by CpG methylation BspOI (BmtI) GCTAGC NheI Overlapping CpG methylation may influence DNA cleavage GCTAGC CpG methylation may influence DNA cleavage Cfr9I (XmaI) CCCGGG SmaI Blocked by CpG methylation CCCGGG Not influenced by CpG methylation Csp6I (CviQI) GTAC RsaI Overlapping CpG methylation may influence DNA cleavage GTAC Overlapping CpG methylation may influence DNA cleavage Ecl136II (EcoICRI) GAGCTC SacI Not influenced by CpG methylation GAGCTC EcoRII CCWGG Blocked by Dcm methylation Not influenced by Dcm methylation MvaI (BstNI) CCWGG HpaII Blocked by CpG methylation CCGG Not influenced by CpG methylation MspI (HpaII) CCGG
References 1. Luria, S.E., Human, M.L., A nonhereditary, host-induced variation of bacterial viruses, J. Bacteriol., 64, 557-569, 1952. 2. Arber, W., Dussoix, D., Host specifi city of DNA producted by Escherichia Coli: I. Host controlled modifi cation of bacteriophage lambda, J. Mol. Biol., 5, 18-36, 1962. 3. Roberts, R.J., et al., A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes, Nucleic Acids Res., 31, 1805-1812, 2003. 4. McClelland, M., The effect of sequence specific DNA methylation on restriction endonuclease cleavage, Nucleic Acids Res., 9, 5859-5866, 1981. 5. McClelland, M., et al., Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases, Nucleic Acids Res., 22, 3640-3659, 1994. 6. Marinus, M.G., Morris, N.R., Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12, J. Bacteriol., 114, 1143-1150, 1973. 7. May, M.S., Hattman, S., Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes, J. Bacteriol., 123, 768-770, 1975. 8. Hattman, S., et al., Sequence specificity of the P1 modification methylase (M.EcoP1) and the DNA methylase (M.Ecodam) controlled by the Escherichia coli dam gene, J. Mol. Biol.,126, 367-380, 1978. 9. Geier, G.E., Modrich, P., Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease, J. Biol. Chem., 254, 1408-1413, 1979. 10. Buryanov, Ya.I., et al., Site specificity and chromatographic properties of E.coli K12 and EcoRII DNA-cytosine methylases, FEBS Letters, 88, 251-254, 1978. 11. Waalwijk, C., Flavell, R.A., MspI, an isochizomer of HpaII which cleaves both unmethylated and methylated HpaII sites, Nucleic Acids Res., 5, 3231-3236, 1978. 12. Bird, A.P., et al., Methylated and unmethylated DNA compartments in the sea urchin genome, Cell, 17, 889-902, 1979. 13. McClelland, M., The frequency and distribution of methylatable DNA sequences in leguminous plant protein coding genes, J. Mol. Evol., 19, 346-354, 1983. 14. Dreiseikelmann, B., et al., The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis, Biochim. Biophys. Acta, 562, 418-428, 1979. 15. Butkus, V. et al., Investigation of restriction-modification enzymes from M.varians RFL19 with a new type of specificity toward modification of substrate, Nucleic Acids Res., 13, 5727-5746, 1985.
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Effect of Dam Methylation on DNA Cleavage

To cleave with a restriction enzyme which is sensitive to the Dam methylation, DNA should be purified from dam E.coli strains. E.coli GM2163 dam, dcm (#M0099) is available upon request with any product purchase. Control digestions should be performed with Lambda DNA (dam, dcm), #SD0021.
Table 1.13. Completely overlapping Dam methyltransferase and restriction enzyme recognition sites. Conventional FastDigest BamHI BamHI BclI BclI BglII BglII Bsp143I Sau3AI BspPI DpnI** DpnI MboI MboI PsuI PsuI PvuI PvuI SfaAI AsiSI Sequence* GGm6ATCC TGm6ATCA AGm6ATCT Gm6ATC GGm6ATC Gm6ATC Gm6ATC RGm6ATCY CGm6ATCG GCGm6ATCGC Effect Table 1.14. Partially overlapping Dam methyltransferase and restriction enzyme recognition sites. Conventional Sequence* Effect FastDigest 5...GAAC(N)4Gm6A TCC...3 AloI 3...CTTG(N)4C Tm6AGG...5 ? 5...TGm6A TC(N)4TCA...3 BdaI Tm6AG(N)4AGT...5 3...AC 5...Gm6A TC(N)3 ATC...3 BseJI 3...C Tm6AG(N)3 TAG...5 BsaBI Bsh1285I 5...CGm6A TCG...3 3...GC Tm6AGC...5 BsiEI 5...Gm6A TCGCGm6A TC...3 Bsp68I 3...C Tm6AGCGC Tm6AG...5 NruI 5...ATCGm6A TC...3 Bsu15I 3...TAGC Tm6AG...5 ClaI 5...Gm6A TC(N)4 VTC...3 Tm6AG(N)4 BAG...5 3...C Hin4I 5...GAY(N)4 Gm6A TC...3 3...CTR(N)4 C Tm6AG...5 5...GGTGm6A TC...3 HphI 3...CCAC Tm6AG...5 5...TCCGGm6A TC...3 3...AGGCC Tm6AG...5 Kpn2I Kpn2I 5...Gm6A TCCGGm6A TC...3 ? 3...C Tm6AGGCC Tm6AG...5 5...GAAGm6A TC...3 MboII 3...CTTC Tm6AG...5 MboII 5...TCATGm6A TC...3 3...AGTAC Tm6AG...5 PagI BspHI 5...Gm6A TCATGm6A TC...3 ? 3...C Tm6AGTAC Tm6AG...5 Note 5...TCCNGGm6A TC...3 PfoI ** DpnI (Gm6ATC) cleaves only Dam 3...AGGNCC Tm6AG...5 PfoI methylated DNA. 5...CTYRAGm6A TC...3 SmoI 3...GARYTC Tm6AG...5 * recognition sequence is indicated in bold. 5...TCGm6A TC...3 TaqI m6A = N6-methyladenine. 3...AGC Tm6AG...5 TaqI overlapping methylase sequences. 5...CAC(N)4Gm6A TCC...3 TstI cleavage not blocked. 3...GTC(N)4C Tm6AGG...5 cleavage blocked. 5...TCTAGm6A TC...5 XbaI 3...AGATC Tm6AG...5 XbaI cleavage rate is slowed significantly by methylation. ? the sensitivity to methylation has not been determined.
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Effect of Dcm Methylation on DNA Cleavage
To cleave with a restriction enzyme which is sensitive to Dcm methylation, DNA should be purified from dcm E.coli strains. E.coli GM2163 dam, dcm (#M0099) is available upon request with any product purchase. Control digestions should be performed with Lambda DNA (dam, dcm), #SD0021.
Table 1.15. Completely overlapping Dcm methyltransferase and restriction enzyme recognition sites. Conventional FastDigest EcoRII MvaI MvaI PasI SexAI SgeI** Sequence* Cm5CWGG Cm5CWGG CCm5CWGGG ACm5CWGGT Cm5CWGG Effect
Table 1.16. Partially overlapping Dcm methyltransferase and restriction enzyme recognition sites. Conventional Sequence* Effect FastDigest 5...GGTACm5CW GG...3 3...CCATG GWm5CC...5 Acc65I Acc65I 5...Cm5CW GGTACm5CW GG...3 3...G GWm5CCATG GWm5CC...5 5...GAAC(N)6 TCm5CW GG...3 AloI 3...CTTG(N)6 AG GWm5CC...5 5...GGGCCm5CW GG...3 ApaI 3...CCCGG GWm5CC...5 ApaI 5...Cm5CW GGATCm5CW GG...3 BamHI 3...G GWm5CCTAG GWm5CC...5 BamHI 5...GTATCm5CW GG...3 BfuI GWm5CC...5 3...CATAG 5...GCm5CW GGNNGGC...3 BglI GWm5CCNNCCG...5 3...CG BglI Bme1390I 5...Cm5CW GG...3 GWm5CC...5 3...G ScrFI 5...Cm5CW GGG...3 BseDI 3...G GWm5CCC...5 BsaJI 5...Cm5CW GGATG...3 BseGI 3...G GWm5CCTAC...5 BseGI 5...Cm5CW GG(N)4 GG...3 3...G GWm5CC(N)4 CC...5 BseLI BslI 5...Cm5CW GGNCm5CW GG...3 3...G GWm5CCNG GWm5CC...5 5...GKGCCm5CW GG...3 BseSI Bme1580I 3...CMCGG GWm5CC...5 5...GGYRCm5CW GG...3 GWm5CC...5 3...CCRYG BshNI BanI 5...Cm5CW GGYRCm5CW GG...3 GWm5CCRYG GWm5CC...5 3...G 5...GGGCCm5CW GG...3 Bsp120I GWm5CC...5 3...CCCGG Bsp120I 5...GGNNCm5CW GG...3 GWm5CC...5 3...CCNNG BspLI NlaIV 5...Cm5CW GGNNCm5CW GG...3 3...G GWm5CCNNG GWm5CC...5 5...Cm5CW GGATC...3 BspPI 3...G GWm5CCTAG...5 5...Cm5CA GG(N)4 TGG...3 3...G GTm5CC(N)4 ACC...5 BstXI BstXI 5...Cm5CA GGNNCm5CT GG...3 3...G GTm5CCNNG GAm5CC...5 5...GGCm5CW GG...3 3...CCG GWm5CC...5 BsuRI HaeIII 5...Cm5CW GGCm5CW GG...3 3...G GWm5CCG GWm5CC...5 5...CAGNNCm5CT GG...3 CaiI 3...GTCNNG GTm5CC...5 AlwNI 5...YGGCm5CW GG...3 CfrI 3...RCCG GWm5CC...5 5...GGNCm5CW GG...3 Cfr13I GWm5CC...5 3...CCNG Sau96I 5...Cm5CW GGGCCm5CW GG...3 Eco24I 3...G GWm5CCCGG GWm5CC...5 Conventional Sequence* Effect FastDigest 5...Cm5CW GGTCTC...3 Eco31I GWm5CCAGAG...5 3...G Eco31I 5...GGWCm5CW GG...3 Eco47I GWm5CC...5 3...CCWG AvaII 5...Cm5CW GGTNACm5CW GG...3 Eco91I 3...G GWm5CCANTG GWm5CC...5 Eco91I 5...Cm5CT GGAG...3 Eco57MI 3...G GAm5CCTC...5 5...AGGCm5CT GG...3 Eco147I 3...TCCG GAm5CC...5 StuI EcoO109I 5...RGGNCm5CT GG...3 EcoO109I 3...YCCNG GAm5CC...5 5...Cm5CW GGCGCm5CW GG...3 EheI 3...G GWm5CCGCG GWm5CC...5 EheI 5...Cm5CW GGGAC...3 FaqI 3...G GWm5CCCTG...5 BsmFI 5...Cm5CW GGATG...3 FokI 3...G GWm5CCTAC...5 5...CTCm5CA GG...3 GsuI GTm5CC...5 3...GAG BpmI 5...GRCGCm5CW GG...3 Hin1I 3...CYGCG GWm5CC...5 BsaHI 5...Cm5CW GGTGA...3 HphI 3...G GWm5CCACT...5 5...GGTACm5CW GG...3 3...CCATG GWm5CC...5 KpnI KpnI 5...Cm5CW GGTACm5CW GG...3 3...G GWm5CCATG GWm5CC...5 5...TGGCm5CA GG...3 MlsI GTm5CC...5 3...ACCG MscI 5...TCm5CA GGA...3 PfoI GTm5CCT...5 3...AG PfoI 5...RGGWCm5CT GG...3 Psp5II GAm5CC...5 3...YCCWG PpuMI 5...Cm5CW GGGCCm5CW GG...3 SduI 3...G GWm5CCCGG GWm5CC...5 Bsp1286I 5...GGCm5CW GGNNGGCC...3 3...CCG GWm5CCNNCCGG...5 SfiI SfiI 5...Cm5CW GGCC (N) GG...3 5GGCm5CW SgsI AscI SspDI TsoI TstI Van91I PflMI XagI EcoNI GGCGCGCm5CW GG...3 GWm5CCGCGCG GWm5CC...5 5...Cm5CW GGCGCC...3 3...G GWm5CCGCGG...5 5...TARCm5CA GG...3 3...ATYG GTm5CC...5 5...CAC(N)6 TCm5CW GG...3 3...GTG(N)6 AG GWm5CC...5 5...Cm5CA GG(N)3 TGG...3 3...G GTm5CC(N)3 ACC...5 5...Cm5CT GG(N)3 AGG...3 3...G GAm5CC(N)3 TCC...5
3...G 5...Cm5CT 3...G 5...Cm5CW 3...G
Note
* recognition sequence is indicated in bold. ** SgeI cleaves only methylated DNA. overlapping methylase sequences. m5C = 5-methylcytosine. cleavage not blocked. cleavage blocked. cleavage rate is slowed significantly by methylation.
GWm5CCGG(N) GWm5CC...5 5CCG
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GGNCm5CA GG...3 GAm5CCNG GTm5CC...5
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Effect of CpG Methylation on DNA Cleavage

Methylated DNA substrates were prepared using SssI methyltransferase.
Table 1.17. CpG is located inside the recognition site. Conventional Sequence FastDigest AatII GACGTC AatII AjiI BauI BcnI NciI Bsh1236I Bsh1236I Bsh1285I BsiEI BshTI AgeI Bsp68I NruI Bsp119I Bsp119I Bsu15I ClaI Cfr9I Cfr10I BsrFI Cfr42I CpoI RsrII CseI HgaI Eco47III AfeI Eco52I EagI Eco72I PmlI Eco88I AvaI Eco105I SnaBI EheI EheI Esp3I BsmBI FspAI FspAI HaeII HhaI HhaI Hin1I BsaHI Hin6I HinP1I CACGTC CACGAG CCSGG CGCG CGRYCG ACCGGT TCGCGA TTCGAA ATCGAT CCCGGG RCCGGY CCGCGG CGGWCCG GACGC AGCGCT CGGCCG CACGTG CYCGRG TACGTA GGCGCC CGTCTC RTGCGCAY RGCGCY GCGC GRCGYC GCGC Effect Conventional FastDigest HpaII HpaII Kpn2I Kpn2I MauBI MauBI MbiI BsrBI MluI MluI MreI MreI MspI MspI NotI NotI NsbI FspI PauI BssHII PdiI NaeI Pfl23II BsiWI Ppu21I BsaAI Psp1406I AclI PvuI PvuI SalI SalI SfaAI AsiSI SgrDI SgsI AscI SmaI SmaI SmuI SsiI AciI SspDI TaiI TaiI TaqI TaqI TauI XhoI XhoI Sequence CCGG TCCGGA CGCGCGCG GAGCGG ACGCGT CGCCGGCG CCGG GCGGCCGC TGCGCA GCGCGC GCCGGC CGTACG YACGTR AACGTT CGATCG GTCGAC GCGATCGC CGTCGACG GGCGCGCC CCCGGG CCCGC CCGC GGCGCC ACGT TCGA GCSGC CTCGAG Effect
Note
cleavage not blocked. cleavage blocked. cleavage rate is reduced significantly by methylation.
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Table 1.18. CpG partially overlaps the recognition site. Conventional Sequence* Effect FastDigest 5...CACCTGm5C G...3 AarI 3...GTGGAC Gm5C...5 5...m5C GAC(N)6GTC...3 3... Gm5CTG(N)6CAG...5 AasI DrdI
5...m5C 3...
Conventional Sequence* Effect FastDigest 5...GAm5C G(N)3GTC...3 Gm5C(N)3CAG...5 3...CT BoxI PshAI
5...GAm5C 3...CT
GAC(N)6 GTm5C G...3 Gm5CTG(N)6 CA Gm5C...5 ? G(N)5GTC...3 Gm5C(N)5CAG...5 BpiI BbsI BplI BplI Bpu10I Bpu10I Bpu1102I BlpI BseDI BsaJI BseGI BseGI BseJI BsaBI
5...GAC(N)4 GTm5C 3...CTG(N)4 CA
5...GAm5C 3...CT 5...GAm5C
Acc65I Acc65I
AdeI DraIII AjuI AjuI AlfI
GGTACm5C G...3 Gm5CCATG Gm5C...5 5...CAm5C GNNGTG...3 3...GT Gm5CNNCAC...5

3... 5...CAm5C 3...GT
5...m5C
G(N)4 m5C GTC...3 ? Gm5CAG...5 3...CT Gm5C(N)4 5...GGTACm5C G...3 Gm5C...5 3...CCATG
5...m5C 3...
G...3 Gm5C...5 5...GAAGAm5C G...3 3...CTTCT Gm5C...5
GNNm5C GTC...3 Gm5CNN Gm5CAG...5
AloI
5...GAAC(N)6 TCm5C 3...CTTG(N)6 AG 5...GAAm5C
GNm5C GTG...3 Gm5CN Gm5CAC...5 5...m5C GAA(N)7TTGG...3 Gm5CTT(N)7AACC...5 3... 5...GCA(N)6TGm5C G...3 Gm5C...5 3...CGT(N)6AC 5...m5C GAAC(N)6TCC...3 3... Gm5CTTG(N)6AGG...5 G...3 Gm5C...5
GAAGAC...3 Gm5CTTCTG...5 5...m5C GAG(N)5 CTm5C G...3 3... Gm5CTC(N)5 GA Gm5C...5 5...CCTNAGm5C G...3 3...GGANTC Gm5C...5 5...m5C GCTNAGm5C G...3 3... Gm5CGANTC Gm5C...5 5...Cm5C Gm5C GG...3 Gm5C Gm5CC...5 3...G 5...m5C GGATG...3 3... Gm5CCTAC...5 5...GAT(N)4 ATm5C G...3 3...CTA(N)4 TA Gm5C...5
3... 5...m5C
Conventional Sequence* Effect FastDigest 5...GTATAm5C G...3 Bst1107I 3...CATAT Gm5C...5 BstZ17I 5...m5C GTATAm5C G...3 3... Gm5CATAT Gm5C...5 5...m5C GGCm5C G...3 BsuRI 3... Gm5CCG Gm5C...5 HaeIII 5...ACCTGm5C G...3 BveI 3...TGGAC Gm5C...5 BspMI 5...YGGCm5C G...3 CfrI Gm5C...5 3...RCCG 5...GGNCm5C G...3 Cfr13I 3...CCNG Gm5C...5 Sau96I 5...m5C GTAm5C G...3 Csp6I 3... Gm5CAT Gm5C...5 Csp6I 5...Gm6A Tm5C G...3 DpnI 3...C Tm6A Gm5C...5 DpnI Eam1104I 5...CTCTTm5C G...3 3...GAGAA Gm5C...5 EarI 5...GAm5C G(N)3 m5C GTC...3 3...CT Gm5C(N)3 Gm5CAG...5 Eam1105I Eam1105I
3...CTG(N)5 CA 3... 5...m5C 5...GAC(N)5 GTm5C
Alw21I Alw21I Alw26I Alw26I Alw44I ApaLI ApaI ApaI BamHI BamHI BfuI
5...m5C 3...
G(N)5TCC...3 3...CTT Gm5C(N)5AGG...5 5...m5C GWGCWm5C G...3 3... Gm5CWCGW Gm5C...5 5...GTCTm5C G...3 3...CAGA Gm5C...5 GTCTC...3 Gm5CAGAG...5 5...GTGCAm5C G...3 3...CACGT Gm5C...5 5...GGGCCm5C G...3 3...CCCGG Gm5C...5
BseLI BslI
G(N)5m5C GG...3 Gm5C(N)5 Gm5CC...5 3...G 5...m5C GCAATG...3 BseMI Gm5CGTTAC...5 3... BsrDI 5...m5C GKGCMm5C G...3 BseSI Bme1580I 3... Gm5CMCGK Gm5C...5 5...m5C GCAGm5C G...3 BseXI Gm5CGTC Gm5C...5 3... BseXI 5...GGYRCm5C G...3 Gm5C...5 3...CCRYG
5...Cm5C
GAT(N)4 ATm5C G...3 Gm5CTA(N)4 TA Gm5C...5 ? 5...Cm5C G(N)6GG...3 3...G Gm5C(N)6CC...5
Ecl136II Ecl136II Eco24I Eco31I Eco31I Eco32I EcoRV Eco47I AvaII Eco91I Eco91I EcoRI EcoRI
5...m5C 3...
5...m5C 3...
GAGCTm5C G...3 Gm5CTCGA Gm5C...5 5...m5C GRGCYm5C G...3 3... Gm5CYCGR Gm5C...5 5...GGTCTm5C G...3 3...CCAGA Gm5C...5
GAC(N)5 GTm5C G...3 Gm5CTG(N)5 CA Gm5C...5 ? 5...GAGCTm5C G...3 3...CTCGA Gm5C...5
G...3 Gm5C...5
BshNI BanI
5...m5C 3...
5...m5C 3...
GGGCCm5C G...3 Gm5CCCGG Gm5C...5 5...m5C GGATCm5C G...3 3... Gm5CCTAG Gm5C...5 5...m5C GTATCC...3 3... Gm5CATAGG...5
5...GTATCm5C 3...CATAG
GGYRCm5C G...3 Gm5CCRYG Gm5C...5
Bsp120I Bsp120I Bsp143I Sau3AI BspLI NlaIV
BglI BglI
G...3 Gm5C...5 5...GCm5C G(N)3 m5C GGC...3 3...CG Gm5C(N)3 Gm5CCG...5

3...CGG(N)5 CC 3... 5...m5C 5...GCC(N)5 GGm5C
GCC...3 Gm5CGG...5 5...GGGCCm5C G...3 3...CCCGG Gm5C...5 5...GATm5C G...3 3...CTA Gm5C...5 5...GGNNCm5C G...3 3...CCNNG Gm5C...5
3...CC 5...m5C
5...GGm5C
GGTCTC...3 Gm5CCAGAG...5 5...m5C GATATm5C G...3 3... Gm5CTATA Gm5C...5 5...GGWCm5C G...3 3...CCWG Gm5C...5 5...m5C GGTNACm5C G...3 3... Gm5CCANTG Gm5C...5 5...m5C GAATTC...3 3... Gm5CTTAAG...5
3...
Bme1390I ScrFI
GCC(N)5GGm5C G...3 Gm5CGG(N)5CC Gm5C...5 5...Cm5C GGG...3 3...G Gm5CCC...5
G...3 Gm5C...5
BspOI BmtI
5...m5C 3...
GGNNCm5C G...3 3... Gm5CCNNG Gm5C...5 5...GCTAGm5C G...3 3...CGATC Gm5C...5 GCTAGm5C G...3 Gm5CGATC Gm5C...5 5...GGATm5C G...3 3...CCTA Gm5C...5 GGATC...3 Gm5CCTAG...5
FaqI BsmFI FokI
5...m5C 3...
GAATTm5C G...3 Gm5CTTAA Gm5C...5 5...GGGAm5C G...3 3...CCCT Gm5C...5
5...m5C
GGGAC...3 Gm5CCCTG...5 5...m5C GGATG...3 3... Gm5CCTAC...5 5...m5C GAY(N)5VTC...3 3... Gm5CTR(N)5BAG...5
5...GAY(N)5 VTm5C 3...CTR(N)5 BA 3... 5...m5C
Hin4I
BspPI
5...m5C 3...
GAY(N)5 VTm5C G...3 Gm5CTR(N)5 BA Gm5C...5?
G...3 Gm5C...5
179
Table 1.18.CpG partially overlaps the recognition site.
1
Conventional Sequence* FastDigest 5...GTYRAm5C G...3 3...CARYT Gm5C...5 HincII HincII

5...m5C 3...
Effect
GTYRAm5C G...3 Gm5CARYT Gm5C...5
5...GTm5C 3...CA
HinfI HinfI HphI Hpy8I Hpy8I
GANTm5C G...3 3... Gm5CTNA Gm5C...5 5...m5C GGTGA...3 Gm5CCACT...5 3... 5...GTNNAm5C G...3 Gm5C...5 3...CANNT
5...m5C 3...
5...m5C
GAC...3 Gm5CTG...5 5...GANTm5C G...3 3...CTNA Gm5C...5
GTNNAm5C G...3 Gm5CANNT Gm5C...5 5...GC(N)7 Gm5C G...3 Gm5C...5 3...CG(N)7 C

5...m5C 3...
HpyF10VI HpyF10VI
GC(N)7Gm5C G...3 Gm5CG(N)7C Gm5C...5 G(N)6GC...3 Gm5C(N)6CG...5
Conventional Sequence* Effect FastDigest 5...GTCAm5C G...3 Gm5C...5 3...CAGT NmuCI NmuCI 5...m5C GTCAm5C G...3 3... Gm5CAGT Gm5C...5 5...CAm5C GNNNGTG...3 Gm5CNNNCAC...5 3...GT OliI AleI 5...CAm5C GNNm5C GTG...3 3...GT Gm5CNN Gm5CAC...5 5...m5C GCATGm5C G...3 PaeI Gm5CGTAC Gm5C...5 3... SphI 5...GAA(N)4 TTm5C G...3 3...CTT(N)4 AA Gm5C...5 PdmI PdmI 5...m5C GAA(N)4 TTm5C G...3 Gm5CTT(N)4 AA Gm5C...5 3... 5...GAWTm5C G...3 PfeI 3...CTWA Gm5C...5 TfiI 5...TCm5C GGGA...3 PfoI 3...AG Gm5CCCT...5 PfoI 5...m5C GAAC(N)5CTC...3 3... Gm5CTTG(N)5GAG...5 PpiI
5...GAAC(N)5 CTm5C 3...CTTG(N)5 GA 5...GAAm5C
Conventional Sequence* Effect FastDigest 5...m5C GGCC(N) G...3 5GGCm5C 3... Gm5CCGG(N) Gm5C...5 5CCG SfiI SfiI
5...GGCm5C 3...CCG 3...CCG 5...GGCm5C
SgeI**
G(N)3m5C GGCC...3 Gm5C(N)3 Gm5CCGG...5 5...m5C GNG...3 3... Gm5CNC...5

5...m5C 3...
G(N)4GGCC...3 Gm5C(N)4CCGG...5
SmoI TaaI TaaI TstI XapI XapI XceI NspI
Gm5C G...3 Gm5C Gm5C...5 5...CTm5C GAG...3 Gm5CTC...5 3...GA 5...Am5C GGT...3 Gm5CCA...5 3...T 5...CAm5C G(N)5TCC...3 Gm5C(N)5AGG...5 3...GT
5...CAC(N)5TCm5C 3...GTG(N)5AG
5...Gm5C 3...C 5...Gm5C
KpnI KpnI KspAI HpaI
LguI SapI Lsp1109I BbvI LweI SfaNI MbiI BsrBI MboI MboI MboII MboII MnlI MnlI MssI MssI
GTTAAm5C G...3 Gm5CAATT Gm5C...5 5...m5C GCTCTTC...3 3... Gm5CGAGAAG...5

3...
5...m5C
G(N)5m5C GC...3 3...C Gm5C(N)5 Gm5CG...5 5...m5C GGTACm5C G...3 3... Gm5CCATG Gm5C...5 5...GTTAAm5C G...3 Gm5C...5 3...CAATT
?
PspFI PsuI PsuI PsyI PsyI RsaI RsaI RseI MslI SacI SacI SanDI
5...m5C
G...3 3...CGAGAA Gm5C...5 5...m5C GCAGm5C G...3 Gm5CGTC Gm5C...5 3... 5...GCATm5C G...3 3...CGTA Gm5C...5 5...m5C GAGCGG...3 3... Gm5CTCGCC...5 5...m5C GATm5C G...3 3... Gm5CTA Gm5C...5 5...m5C GAAGA...3 3... Gm5CTTCT...5 5...CCTm5C G...3 3...GGA Gm5C...5 5...GTTTAAAm5C G...3 3...CAAATTT Gm5C...5
5...GCTCTTm5C
5...m5C 3...
5...m5C 3...
GTAm5C G...3 Gm5CAT Gm5C...5 5...CAm5C GNNNRTG...3 Gm5CNNNYAC...5 3...GT 5...m5C GAGCTm5C G...3 Gm5CTCGA Gm5C...5 3... 5...GGGWCCm5C G...3 3...CCCWGG Gm5C...5
G(N)4CTC...3 3...CTT Gm5C(N)4GAG...5 5...CCCAGm5C G...3 3...GGGTC Gm5C...5 5...m5C GGATCm5C G...3 3... Gm5CCTAG Gm5C...5 5...GAm5C GNNGTm5C G...3 3...CT Gm5CNNCA Gm5C...5 5...GTAm5C G...3 3...CAT Gm5C...5
G...3 Gm5C...5
5...m5C 3...
G...3 Gm5C...5 5...m5C GAATTm5C G...3 Gm5CTTAA Gm5C...5 3... 5...RCATGm5C G...3 3...YGTAC Gm5C...5 GCATGm5C G...3 Gm5CGTAC Gm5C...5 5...GTm5C GAC...3 3...CA Gm5CTG...5
5...GTMKAm5C 3...CAKMT
XmiI AccI
G...3 Gm5C...5
SatI Fnu4HI
5...GCNGm5C 3...CGNC
GGGWCCm5C G...3 Gm5CCWGG Gm5C...5 5...Gm5C GGC...3 3...C Gm5CCG...5 G...3 Gm5C...5 5...GAGTm5C G...3 3...CTCA Gm5C...5
Note
* recognition sequence is indicated in bold. ** SgeI cleaves only methylated DNA. overlapping methylase sequences. m5C = 5-methylcytosine. cleavage blocked. cleavage rate is reduced significantly by methylation. ? the sensitivity to methylation has not been determined. cleavage not blocked.
Mva1269I Mva1269I
5...m5C 3...
GTTTAAAm5C G...3 3... Gm5CAAATTT Gm5C...5 5...GAATGm5C G...3 3...CTTAC Gm5C...5 GAATGC...3 Gm5CTTACG...5 5...GCTAGm5C G...3 3...CGATC Gm5C...5
SchI MlyI SduI Bsp1286I SfaNI
5...m5C
GAGTC...3 3... Gm5CTCAG...5 5...m5C GDGCHm5C G...3 3... Gm5CHCGD Gm5C...5 5...m5C GCATC...3 3... Gm5CGTAG...5
5...GCATm5C 3...CGTA
Single letter code

NheI NheI
5...m5C 3...
GCTAGm5C G...3 Gm5CGATC Gm5C...5
G...3 Gm5C...5
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Effect of EcoKI and EcoBI Methylation on DNA Cleavage
Methylated DNA substrates were purified from E.coli K12 or E.coli B strains.
Table 1.19. EcoBI overlapping methylation. Conventional Sequence* Effect FastDigest AatII 5...TGm6ACGTC(N)4 TGCT...3 AatII TGCAG(N)4m6ACGA...5 3...AC AluI 5...TGm6AGCT(N)5 TGCT...3 AluI 3...AC TCGA(N)5m6ACGA...5 BclI 5...TGm6A TCA(N)5 TGCT...3 BclI 3...AC Tm6AGT(N)5m6ACGA...5 BglII 5...TGm6AGATCT(N)3 TGCT...3 BglII TCTAGA(N)3m6ACGA...5 3...AC Bpu10I 5...CCTGm6AGC(N)6 TGCT...3 BpuI TCG(N)6m6ACGA...5 3...GGAC Bsp143I 5...TGm6ATC(N)6 TGCT...3 Sau3AI 3...AC TAG(N)6 m6ACGA...5 EcoRI 5...TGm6AATTC(N)4 TGCT...3 EcoRI TTAAG(N)4m6ACGA...5 3...AC HincII HincII
3...CAAC 3...AC 5...GTTGm6AC(N)7
Table 1.20. EcoKI overlapping methylation. Conventional Sequence* Effect FastDigest Alw21I 5...Am6AC(N)6 G TGCWC...3 Alw21I 3...T TG(N)6Cm6ACGWG...5 Alw44I 5...Am6AC(N)6 G TGCAC...3 ApaLI 3...T TG(N)6Cm6ACGTG...5 BseSI 5...Am6AC(N)6 G TGCMC...3 Bme1580I 3...T TG(N)6Cm6ACGKG...5 DraI 5...TTTAAm6AC(N)6 G TGC...3 DraI TG(N)6Cm6ACG...5 3...AAATT HincII 5...GTYAm6AC(N)6 G TGC...3 HincII 3...CART TG(N)6Cm6ACG...5 KspAI 5...GTTAm6AC(N)6 G TGC...3 HpaI 3...CAAT TG(N)6Cm6ACG...5 MluI 5...Am6ACGCGTNNG TGC...3 MluI TGCGCANNCm6ACG...5 3...T MssI 5...GTTTAAm6AC(N)6 G TGC...3 MssI 3...CAAATT TG(N)6Cm6ACG...5 5...Am6AC(N)6 G TGCGCA...3 NsbI 3...T TG(N)6Cm6ACGCGT...5 FspI OliI AleI
5...Am6ACAC(N)4 G 3...T
HindIII HindIII HinfI HinfI HphI MboI MboI MboII MboII MluI MluI MnlI MnlI NdeI NdeI OliI AleI PaeI SphI PagI BspHI SacI SacI ScaI ScaI SspI SspI TasI Tsp509I
5...TGm6AAGCTT(N)3 5...TGm6ANTC(N)5 5...GGTGm6A(N)8
TGCT...3 TTCGAA(N)3m6ACGA...5 TGCT...3 TNAG(N)5m6ACGA...5 TGCT...3 T(N)8m6ACGA...5
TGCT...3 TG(N)7m6ACGA...5
Recognition sequences of the following endonucleases may also overlap with DNA sequences methylated by EcoBI and EcoKI. The following conventional restriction enzymes and the respective FastDigest enzymes have not been tested for sensitivity to EcoBI methylation: AarI, AdeI, AjiI, Alw21I, BauI, BcuI, BfiI, BglII, BoxI, BpiI, Bpu1102I, BseGI, BseJI, BseMI, BseNI, BseXI, BshTI, Bsp143II, BspPI, Bsu15I, BveI, CaiI, Cfr10I, CseI, DpnI, Eam1104I, Ecl136II, Eco24I, Eco31I, Eco32I, Eco47III, Eco57I, Eco57MI, Eco72I, Eco81I, Eco91I, Eco147I, EcoO109I, Esp3I, FspAI, FokI*, HaeII*, HindIII,Hin1I, Hin1II, Hin4I, Hpy8I, HpyF3I, LguI, Lsp1109I, LweI, MbiI, Mph1103I, MunI, Mva1269I, NdeI, NmuCI, PdmI, PfeI, Ppu21I, PscI, Psp5II, Psp1406I, PsuI, PsyI, PvuII, RseI, SchI, SduI, SexAI*, SfaNI*, SmiI, SmoI, SspI, TaaI, TaiI, TatI, TscAI, TstI, VspI, XagI, XapI and XceI. The following conventional restriction enzymes and the respective FastDigest enzymes have not been tested for sensitivity to EcoKI methylation: AanI, AdeI, AjiI, BauI, BcuI, BfiI, BseNI, BshNI, BshTI, Bsp119I, BveI, Cfr10I, Eco72I, Eco91I, FspAI, Hpy8I, MseI*, PacI, PdmI, Ppu21I, PscI, Psp1406I, SexAI*, SduI, TaaI, TaiI, TscAI, TsoI and XceI.
* enzymes are available in FastDigest format only.
3...AC
3...CCAC 3...AC 3...AC 3...AC 3...AC 3...AC
RseI MslI Tru1I Tru1I
5...TGm6ATC(N)6
5...TGm6AAGA(N)5
TGCT...3 TAG(N)6m6ACGA...5
3...CG
5...GCm6ACNNNNRTG 5...TTAm6AC(N)6 G
TGC...3 TGTG(N)4Cm6ACG...5
TT...3 TGNNNNYACm6AT...5 TGC...3 TG(N)6Cm6ACG...5
3...AAT
5...TGm6ACGCGT(N)3 5...TGm6AGG(N)6
TGCT...3 TTCT(N)5m6ACGA...5
TGCT...3 TGCGCA(N)3m6ACGA...5 TGCT...3 TCC(N)6m6ACGA...5
5...TGm6A(N)4CATA
3...AC 3...AC
5...TGm6A(N)4 CACG 5...TGm6A(N)5 GCA 5...TCATGm6A(N)8
TGCT...3 T(N)4GTATm6ACGA...5
TGCT...3 T(N)4GTGCm6ACGA...5 TGCT...3 T(N)5CGTm6ACGA...5 TGCT...3 T(N)8m6ACGA...5
Note
* recognition sequence is indicated in bold. cleavage not blocked. cleavage blocked. cleavage rate is reduced significantly by methylation.
3...AGTAC 3...AC 3...AC 3...AC 3...AC
5...TGm6AGCTC(N)4
5...TGm6AGTACT(N)3 5...TGm6AATATT(N)3 5...TGm6AATT(N)5
TGCT...3 TCGAG(N)4m6ACGA...5
TGCT...3 TCATGA(N)3m6ACGA...5 TGCT...3 TTATAA(N)3m6ACGA...5 TGCT...3 TTAA(N)5m6ACGA...5
Single letter code

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Newly Generated Cleavage Sites

Recognition Sites Resulting from Ligation of Blunt DNA Ends
* Restriction enzymes that have degenerate recognition sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed. Fermentas FastDigest enzymes are shown in ruby. Fermentas conventional enzymes are shown in orange.
Note
Single letter code

Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends.
First restriction enzyme Second restriction enzyme Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC), FaiI* (YATA), FaiI* (YATG) DraI/FastDigest DraI (TTTAAA), Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC), MssI (PmeI)/ FastDigest MssI (GTTTAAAC), RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT) Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) EheI (SfoI)/FastDigest EheI (GGCGCC) SspI/FastDigest SspI (AATATT) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA) AjiI (BmgBI)* (CACGTC) Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG) FaiI* (YATG) MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) ZraI (GACGTC) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) DpnI/FastDigest DpnI (GATC) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/ FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) EheI (SfoI)/FastDigest EheI (GGCGCC) MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) ZraI (GACGTC) AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MlsI (MscI)/FastDigest MscI (MlsI) (TGGCCA) CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) EheI (SfoI)/FastDigest EheI (GGCGCC) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) Bsh1236I (BstUI)/ FastDigest Bsh1236I (CGCG) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) EheI (SfoI)/FastDigest EheI (GGCGCC) Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) Restriction enzymes that cleave the newly generated recognition sequence FaiI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I SetI AluI/FastDigest AluI, CviJI, SetI CviJI FastDigest MseI (SaqAI), TasI (Tsp509I)/FastDigest Tsp509I (TasI), Tru1I (MseI)/FastDigest Tru1I SetI MnlI/FastDigest MnlI, SetI MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest TaiI Eco72I (PmlI)/FastDigest PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI (MaeII)/ FastDigest TaiI TscAI (TspRI)/FastDigest TspRI (TscAI) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BceAI AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest TaiI SetI HinfI/FastDigest HinfI MnlI/FastDigest MnlI, SetI MaeII, SetI, TaiI (MaeII)/FastDigest TaiI CseI (HgaI)/FastDigest HgaI (CseI) CseI (HgaI)/FastDigest HgaI (CseI), Hin1I (BsaHI)/FastDigest BsaHI (Hin1I) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BceAI AatII/FastDigest AatII, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), MaeII, SetI, TaiI (MaeII)/FastDigest TaiI, ZraI SetI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI CviJI AluI/FastDigest AluI, CviJI, SetI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ FastDigest DpnI, MboI/FastDigest MboI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ FastDigest DpnI, MboI/FastDigest MboI CviJI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI Bsh1236I (BstUI)/FastDigest Bsh1236I (continued on next page)
AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA)
AjiI (BmgBI)* (GACGTG)
AluI/FastDigest AluI (AGCT)
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Table 1.21.Newly generated recognition sites resulting from ligation of blunt DNA ends.
First restriction enzyme Second restriction enzyme Restriction enzymes that cleave the newly generated recognition sequence
Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), Bsh1236I (BstUI)/FastDigest Bsh1236I MspA1I* (CMGCGG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) DraI/FastDigest DraI (TTTAAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC), MssI (PmeI)/FastDigest MssI (GTTTAAAC), RsaI/FastDigest RsaI TaqI/FastDigest TaqI Bsp68I (NruI)/Fast- (GTAC), ScaI/FastDigest ScaI (AGTACT), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT), SspI/ FastDigest SspI (AAT ATT) Digest NruI (RruI) (TCGCGA) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ FastDigest DpnI, MboI/FastDigest MboI, TaqI/FastEco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) Digest TaqI CviJI Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) Hpy188I HincII (HindII)/FastDigest HincII* (GTYGAC) AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), FaiI* (YATA), FaiI* (YATG) FaiI AluI/FastDigest AluI (AGCT), Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG), Bsp68I (NruI)/ FastDigest NruI (RruI) (TCGCGA), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), HpyCH4V (TGCA), MbiI (BsrBI)/ Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI FastDigest BsrBI (MbiI)* (CCGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MlsI (MscI)/ FastDigest MscI (MlsI) (TGGCCA), MspA1I* (CMGCGG), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Bst1107I (BstZ17I)/ Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI, FastDigest BstZ17I Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT) SetI (Bst1107I) (GTATAC) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) AluI/FastDigest AluI, CviJI, SetI CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) Hpy8I (MjaIV)/FastDigest Hpy8I HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) Hpy8I (MjaIV)/FastDigest Hpy8I, XmiI (AccI)/FastDigest HincII (HindII)/FastDigest HincII* (GTYGAC) AccI (XmiI) MaeIII RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT) TasI (Tsp509I)/FastDigest Tsp509I (TasI) SspI/FastDigest SspI (AATATT) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* CviJI (CCGCTC), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), MlsI (MscI)/FastDigest MscI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI BsuRI (HaeIII)/FastDi- (MlsI) (TGGCCA) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI gest HaeIII (BsuRI) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) (GGCC) (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), EheI (SfoI)/FastDigest EheI (GGCGCC) Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI FspAI/FastDigest FspAI* (RTGCGCAC) (Bsp1286I)/FastDigest Bsp1286I (SduI) FaqI (BsmFI)/FastDigest BsmFI (FaqI) HincII (HindII)/FastDigest HincII* (GTYGAC) AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI SetI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastAluI/FastDigest AluI, CviJI, SetI Digest BsrBI (MbiI)* (CCGCTC), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) CviJI* (AGCY) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MlsI (MscI)/FastDigest MscI (MlsI) CviJI (TGGCCA) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) AluI/FastDigest AluI (AGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), Eco47III (AfeI)/ FastDigest AfeI (Eco47III) (AGCGCT), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC), MspA1I* CviJI (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI (AGGCCT), MlsI (MscI)/FastDigest MscI (MlsI) (TGGCCA) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI CviJI* (GGCY) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), EheI (SfoI)/FastDigest EheI (GGCGCC) Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI FspAI/FastDigest FspAI* (RTGCGCAC) (Bsp1286I)/FastDigest Bsp1286I (SduI) FaqI (BsmFI)/FastDigest BsmFI (FaqI) HincII (HindII)/FastDigest HincII* (GTYGAC) (continued on next page)
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Restriction enzymes that cleave the newly generated recognition sequence HinfI/FastDigest HinfI, PleI, SchI (MlyI)/FastDigest AjiI (BmgBI)* (CACGTC), ZraI (GACGTC) MlyI (SchI) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) HinfI/FastDigest HinfI SetI Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT) HinfI/FastDigest HinfI, PfeI (TfiI)/FastDigest TfiI (PfeI) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) DpnI/FastDigest DpnI (GATC) AluI/FastDigest AluI, CviJI, SetI Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) Alw21I (BsiHKAI)/FastDigest Alw21I, SduI (Bsp1286I)/ FspAI/FastDigest FspAI* (RTGCGCAC) FastDigest Bsp1286I (SduI) TasI (Tsp509I)/FastDigest Tsp509I (TasI) SspI/FastDigest SspI (AATATT) AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) TaqI/FastDigest TaqI Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) DraI/FastDigest FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDiDraI (TTTAAA) HpyCH4V gest FspI (NsbI) (TGCGCA) DraI/FastDigest DraI, FastDigest MseI (SaqAI), Tru1I MssI (PmeI)/FastDigest MssI (GTTTAAAC), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT) (MseI)/FastDigest Tru1I AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI SetI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII AluI/FastDigest AluI, CviJI, SetI (CAGCTG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MlsI (MscI)/FastDigest CviJI Ecl136II (EcoICRI)/ MscI (MlsI) (TGGCCA) FastDigest Ecl136II HinfI/FastDigest HinfI, PleI, SchI (MlyI)/FastDigest (GAGCTC) DpnI/FastDigest DpnI (GATC) MlyI (SchI) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) AluI/FastDigest AluI, Alw21I (BsiHKAI)/FastDigest Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest Ecl136II, MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Eco24I (BanII), SacI/FastDigest SacI, SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI), SetI AjiI (BmgBI)* (CACGTC), ZraI (GACGTC) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI AjiI (BmgBI)* (GACGTG), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDi- MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, gest BsaAI (Ppu21I)* (YACGTG) TaiI (MaeII)/FastDigest TaiI AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII SetI (CAGCTG) Eco105I (SnaBI)/ FastDigest SnaBI Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI, SetI (Eco105I) (TACGTA) MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BceAI PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA) (MaeII)/FastDigest TaiI AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), DpnI/FastDigest DpnI (GATC), FaiI* (YATA), FaiI* SetI (YATG) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* CviJI (CCGCTC), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI, Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) SetI Eco147I (StuI)/ BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), MlsI (MscI)/FastDigest MscI (MlsI) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI FastDigest StuI (TGGCCA) (Eco147I) (AGGCCT) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), EheI (SfoI)/FastDigest EheI (GGCGCC) Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI FspAI/FastDigest FspAI* (RTGCGCAC) (Bsp1286I)/FastDigest Bsp1286I (SduI) FaqI (BsmFI)/FastDigest BsmFI (FaqI) HincII (HindII)/FastDigest HincII* (GTYGAC) Second restriction enzyme (continued on next page)
First restriction enzyme
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First restriction enzyme Second restriction enzyme Restriction enzymes that cleave the newly generated recognition sequence FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I
AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA) AluI/FastDigest AluI (AGCT), Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), HpyCH4V (TGCA), MbiI (BsrBI)/ Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ FastDigest BsrBI (MbiI)* (CCGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MlsI (MscI)/ FastDigest DpnI, MboI/FastDigest MboI FastDigest MscI (MlsI) (TGGCCA), MspA1I* (CMGCGG), MspA1I* (CMGCTG), PvuII/FastDigest Eco32I (EcoRV)/ PvuII (CAGCTG) FastDigest EcoRV Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI, TaqI/FastBsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Digest TaqI HinfI/FastDigest HinfI, PfeI (TfiI)/FastDigest TfiI (PfeI) DpnI/FastDigest DpnI (GATC) HpyCH4V FspAI/FastDigest FspAI* (RTGCGCAC), NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) FspAI/FastDigest FspAI* (RTGCGCAT) AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) AluI/FastDigest AluI, CviJI, SetI (GTATAC), DpnI/FastDigest DpnI (GATC), FaiI* (YATA), FaiI* (YATG) AluI/FastDigest AluI (AGCT), Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG), Bsp68I (NruI)/ FastDigest NruI (RruI) (TCGCGA), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), CviJI CviJI* (RGCT), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), HpyCH4V (TGCA), MlsI (MscI)/ FastDigest MscI (MlsI) (TGGCCA), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) CviJI, MnlI/FastDigest MnlI LweI (SfaNI)/FastDigest SfaNI (BmsI) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) Eco47III (AfeI)/ FastDigest HaeII (BfoI), HhaI/FastDigest HhaI, Hin6I FastDigest AfeI EheI (SfoI)/FastDigest EheI (GGCGCC) (Eco47III) (AGCGCT) (HinP1I)/FastDigest HinP1I (Hin6I) FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDi- HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest gest FspI (NsbI) (TGCGCA) HinP1I (Hin6I) CviJI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) MspI SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/FastDi PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) gest TauI SrfI (GCCCGGGC) Cac8I AjiI (BmgBI)* (CACGTC), ZraI (GACGTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest TaiI Eco72I (PmlI)/FastDigest PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI (MaeII)/ AjiI (BmgBI)* (GACGTG), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG) FastDigest TaiI AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII SetI Eco72I (PmlI)/FastDi- (CAGCTG) gest PmlI (Eco72I) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI, SetI (CACGTG) Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, (YACGTA) TaiI (MaeII)/FastDigest TaiI FaiI* (YATG) TscAI (TspRI)/FastDigest TspRI (TscAI) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) BceAI PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) CviJI (GTATAC), DpnI/FastDigest DpnI (GATC), FaiI* (YATA), FaiI* (YATG) AjiI (BmgBI)* (CACGTC), ZraI (GACGTC) Hin1I (BsaHI)/FastDigest BsaHI (Hin1I) AluI/FastDigest AluI (AGCT), Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG), Bsp68I (NruI)/ FastDigest NruI (RruI) (TCGCGA), CviJI* (RGCT), HpyCH4V (TGCA), MspA1I* (CMGCTG), PvuII/ BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI FastDigest PvuII (CAGCTG) BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), MlsI (MscI)/FastDigest MscI (MlsI) (TGGCCA) Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, MnlI/ Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) EheI (SfoI)/ FastDigest MnlI FastDigest EheI LweI (SfaNI)/FastDigest SfaNI (BmsI) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) (GGCGCC) FastDigest HaeII (BfoI), HhaI/FastDigest HhaI, Hin6I Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) (HinP1I)/FastDigest HinP1I (Hin6I) FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDi- HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest gest FspI (NsbI) (TGCGCA) HinP1I (Hin6I) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, HpaII/ MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) FastDigest HpaII, MspI (HpaII)/FastDigest MspI SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/FastDiPdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) gest TauI SrfI (GCCCGGGC) Cac8I AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) FaiI (GTATAC) AjiI (BmgBI)* (GACGTG), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), MspA1I* (CMGCTG), TscAI (TspRI)/FastDigest TspRI (TscAI) Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), PvuII/FastDigest PvuII (CAGCTG) FaiI* (CATR) SetI Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT) AluI/FastDigest AluI, CviJI, SetI Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) TasI (Tsp509I)/FastDigest Tsp509I (TasI) SspI/FastDigest SspI (AATATT)
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Second restriction enzyme AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) EheI (SfoI)/FastDigest EheI (GGCGCC) SspI/FastDigest SspI (AATATT) DraI/FastDigest DraI (TTTAAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC), MssI (PmeI)/FastDigest MssI (GTTTAAAC), RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC)
Restriction enzymes that cleave the newly generated recognition sequence FaiI SetI AluI/FastDigest AluI, CviJI, SetI CviJI TasI (Tsp509I)/FastDigest Tsp509I (TasI) HpyCH4VI MnlI/FastDigest MnlI LweI (SfaNI)/FastDigest SfaNI (BmsI), HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I), NsbI (FspI)/FastDigest FspI (NsbI) SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/FastDigest TauI Cac8I HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) Alw21I (BsiHKAI)/FastDigest Alw21I, SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI)
FaiI* (TATR)
FspAI/FastDigest Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), EheI (SfoI)/FastDigest EheI (GGCGCC) FspAI* (ATGCGCAY) MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) SrfI (GCCCGGGC) SspI/FastDigest SspI (AATATT) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), MlsI (MscI)/FastDigest MscI (MlsI) (TGGCCA) DpnI/FastDigest DpnI (GATC)
DraI/FastDigest DraI (TTTAAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC), MssI (PmeI)/FastDigest MssI (GTTTAAAC), SmiI (SwaI)/FastDigest HpyCH4V SwaI (SmiI) (ATTTAAAT), SspI/FastDigest SspI (AATATT) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI LweI (SfaNI)/FastDigest SfaNI (BmsI), HpyCH4V Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), EheI (SfoI)/FastDigest EheI (GGCGCC) HinP1I (Hin6I) FspAI/FastDigest FspAI* (GTGCGCAY) MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) HinP1I (Hin6I), NsbI (FspI)/FastDigest FspI (NsbI) SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/FastDiPdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) gest TauI Alw21I (BsiHKAI)/FastDigest Alw21I, Alw44I (ApaLI)/ FastDigest ApaLI (AIw44I), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT) Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) SrfI (GCCCGGGC) Cac8I Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Hpy188I Hpy8I (MjaIV)/FastDigest Hpy8I, XmiI (AccI)/FastDigest Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) AccI (XmiI) Alw26I (BsmAI)/FastDigest Alw26I DpnI/FastDigest DpnI (GATC) HincII (HindII)/ FastDigest HincII* Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI (GTCRAC) HincII (HindII)/FastDigest HincII, Hpy8I (MjaIV)/FastDiKspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) gest Hpy8I HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) MaeIII, NmuCI (Tsp45I)/FastDigest NmuCI RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT) AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA), DraI/ FastDigest DraI (TTTAAA), MssI (PmeI)/FastDigest MssI (GTTTAAAC), SmiI (SwaI)/FastDigest FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I SwaI (SmiI) (ATTTAAAT) TaqI/FastDigest TaqI Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Hpy8I (MjaIV)/FastDigest Hpy8I Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) HincII (HindII)/ FastDigest HincII* FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDiHpyCH4V (GTTRAC) gest FspI (NsbI) (TGCGCA) HincII (HindII)/FastDigest HincII, Hpy8I (MjaIV)/FastDigest Hpy8I, KspAI (HpaI)/FastDigest HpaI (KspAI), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I MaeIII RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI HpyCH4V (TGCA) CviJI Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) (continued on next page)
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First restriction enzyme Second restriction enzyme Restriction enzymes that cleave the newly generated recognition sequence FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I TaqI/FastDigest TaqI Hpy8I (MjaIV)/FastDigest Hpy8I HpyCH4V HincII (HindII)/FastDigest HincII, Hpy8I (MjaIV)/FastDigest Hpy8I, KspAI (HpaI)/FastDigest HpaI (KspAI), FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I HincII (HindII)/FastDigest HincII, Hpy8I (MjaIV)/FastDigest Hpy8I MaeIII
AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), DraI/FastDigest DraI (TTTAAA), MssI (PmeI)/FastDigest MssI (GTTTAAAC), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT) Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDiKspAI (HpaI)/FastDigest FspI (NsbI) (TGCGCA) gest HpaI (KspAI) (GTTAAC) HincII (HindII)/FastDigest HincII* (GTYAAC) HincII (HindII)/FastDigest HincII* (GTYGAC)
MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC)
MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG)
RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT) AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), HincII (HindII)/FastDigest HincII* (GTYGAC), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), CviJI* (RGCT), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), HpyCH4V (TGCA), MlsI (MscI)/ SsiI (AciI)/FastDigest AciI (SsiI) FastDigest MscI (MlsI) (TGGCCA) Bsh1236I (BstUI)/FastDigest Bsh1236I, SsiI (AciI)/ Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG), Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) FastDigest AciI (SsiI) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) MbiI (BsrBI)/FastDigest BsrBI (MbiI), SsiI (AciI)/FastDiEcl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC) gest AciI (SsiI) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI CviJI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) MspI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, HpaII/ EheI (SfoI)/FastDigest EheI (GGCGCC) FastDigest HpaII, MspI (HpaII)/FastDigest MspI BseDI (BsaJI)/FastDigest BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, MspA1I* (CMGCGG) SsiI (AciI)/FastDigest AciI (SsiI) MspA1I, SsiI (AciI)/FastDigest AciI (SsiI) MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest SmaI/FastDigest SmaI (CCCGGG), SrfI (GCCCGGGC) BsaJI (BseDI), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI SetI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII AluI/FastDigest AluI, CviJI, SetI (CAGCTG) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MlsI (MscI)/FastDigest CviJI MscI (MlsI) (TGGCCA) HinfI/FastDigest HinfI, PleI, SchI (MlyI)/FastDigest DpnI/FastDigest DpnI (GATC) MlyI (SchI) AluI/FastDigest AluI, Alw21I (BsiHKAI)/FastDigest Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest Ecl136II, Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC) Eco24I (BanII), SacI/FastDigest SacI, SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI), SetI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) (continued on next page)
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Second restriction enzyme AluI/FastDigest AluI (AGCT), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC), MspA1I* (CMGCTG), PvuII/FastDigest PvuII (CAGCTG) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) EheI (SfoI)/FastDigest EheI (GGCGCC) FspAI/FastDigest FspAI* (RTGCGCAC) HincII (HindII)/FastDigest HincII* (GTYGAC) AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MlsI (MscI)/FastDigest MscI (MlsI) (TGGCCA) Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) EheI (SfoI)/FastDigest EheI (GGCGCC) FaiI* (YATG) MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG) PvuII/FastDigest PvuII (CAGCTG)
Restriction enzymes that cleave the newly generated recognition sequence CviJI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) FaqI (BsmFI)/FastDigest BsmFI (FaqI) SetI AluI/FastDigest AluI, CviJI, SetI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI CviJI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ FastDigest DpnI, MboI/FastDigest MboI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI TscAI (TspRI)/FastDigest TspRI (TscAI) MspA1I AluI/FastDigest AluI, CviJI, MspA1I, PvuII/FastDigest PvuII, SetI
MlsI (MscI)/FastDigest MscI (MlsI) (TGGCCA)
MspA1I* (CAGCKG)
AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), HincII (HindII)/FastDigest HincII* (GTYGAC), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), CviJI* (RGCT), Eco147I (StuI)/FastDigest StuI (Eco147I) (AGGCCT), HpyCH4V (TGCA), MlsI (MscI)/ SsiI (AciI)/FastDigest AciI (SsiI) FastDigest MscI (MlsI) (TGGCCA) Bsh1236I (BstUI)/FastDigest Bsh1236I, SsiI (AciI)/ Bsh1236I (BstUI)/FastDigest Bsh1236I (CGCG),Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) FastDigest AciI (SsiI) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) MbiI (BsrBI)/FastDigest BsrBI (MbiI), SsiI (AciI)/FastDiEcl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) gest AciI (SsiI) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI MspA1I* (CCGCKG) CviJI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT) MspI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, HpaII/ EheI (SfoI)/FastDigest EheI (GGCGCC) FastDigest HpaII, MspI (HpaII)/FastDigest MspI BseDI (BsaJI)/FastDigest BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG) SsiI (AciI)/FastDigest AciI (SsiI) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) HpaII, MspI (HpaII)/FastDigest MspI MspA1I, SsiI (AciI)/FastDigest AciI (SsiI) PvuII/FastDigest PvuII (CAGCTG) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest SmaI/FastDigest SmaI (CCCGGG), SrfI (GCCCGGGC) BsaJI (BseDI), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I, (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) TaqI/FastDigest TaqI Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) DraI/FastDigest DraI, FastDigest MseI (SaqAI), Tru1I MssI (PmeI)/ DraI/FastDigest DraI (TTTAAA), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT) (MseI)/FastDigest Tru1I FastDigest MssI (GTTTAAAC) FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDiHpyCH4V gest FspI (NsbI) (TGCGCA) RsaI/FastDigest RsaI (GTAC), ScaI/FastDigest ScaI (AGTACT) Hpy8I (MjaIV)/FastDigest Hpy8I (continued on next page)
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DraI/FastDigest DraI (TTTAAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC), MssI (PmeI)/FastDigest MssI (GTTTAAAC), RsaI/FastDigest RsaI HpyCH4V (GTAC), ScaI/FastDigest ScaI (AGTACT), SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT), SspI/ FastDigest SspI (AATATT) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI LweI (SfaNI)/FastDigest SfaNI (BmsI) , HpyCH4V Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) NsbI (FspI)/FastDiHhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), EheI (SfoI)/FastDigest EheI (GGCGCC) gest FspI (NsbI) HinP1I (Hin6I) (TGCGCA) HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT) HinP1I (Hin6I), NsbI (FspI)/FastDigest FspI (NsbI) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/FastDiPdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) gest TauI SrfI (GCCCGGGC) Cac8I DpnI/FastDigest DpnI (GATC), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/ MnlI/FastDigest MnlI FastDigest BsrBI (MbiI)* (CCGCTC) BccI Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) PdiI (NaeI)/FastDiEco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), EheI (SfoI)/FastDigest EheI (GGCGCC), FspAI/ SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDi gest NaeI (PdiI) FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest gest AciI (SsiI), TauI/FastDigest TauI (GCCGGC) FspI (NsbI) (TGCGCA) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG), SmaI/FastDigest SmaI FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest (CCCGGG), SrfI (GCCCGGGC) HpaII, MspI (HpaII)/FastDigest MspI AjiI (BmgBI)* (CACGTC), ZraI (GACGTC) AjiI (BmgBI), MaeII, SetI, TaiI (MaeII)/FastDigest TaiI Eco72I (PmlI)/FastDigest PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI (MaeII)/ AjiI (BmgBI)* (GACGTG), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG) FastDigest TaiI AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII SetI Ppu21I (BsaAI)/ (CAGCTG) FastDigest BsaAI Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI, SetI (Ppu21I)* (CACGTR) MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA) TaiI (MaeII)/FastDigest TaiI FaiI* (YATG) TscAI (TspRI)/FastDigest TspRI (TscAI) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) BceAI PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) AjiI (BmgBI)* (CACGTC), ZraI (GACGTC) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, AjiI (BmgBI)* (GACGTG), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG) TaiI (MaeII)/FastDigest TaiI AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII SetI (CAGCTG) Ppu21I (BsaAI)/ FastDigest BsaAI Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI, SetI (Ppu21I)* (TACGTR) Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA) (MaeII)/FastDigest TaiI HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) BceAI PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) AjiI (BmgBI)* (CACGTC), AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI SetI (Ppu21I)* (YACGTA), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG), ZraI (GACGTC) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), AluI/FastDigest AluI, CviJI, SetI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI) (GGCC), CviJI* (RGCC), Eco147I (StuI)/FastDigest StuI PvuII/FastDigest (Eco147I) (AGGCCT), Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), MlsI (MscI)/FastDigest CviJI MscI (MlsI) (TGGCCA) PvuII (CAGCTG) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/ Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) FastDigest DpnI, MboI/FastDigest MboI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI EheI (SfoI)/FastDigest EheI (GGCGCC) FaiI* (YATG) TscAI (TspRI)/FastDigest TspRI (TscAI) MspA1I MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) AluI/FastDigest AluI, CviJI, MspA1I, PvuII/FastDigest MspA1I* (CMGCTG) PvuII, SetI (continued on next page)
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Second restriction enzyme
RsaI/FastDigest RsaI (GTAC)
ScaI/FastDigest ScaI (AGTACT)
SmaI/FastDigest SmaI (CCCGGG)
SmiI (SwaI)/FastDigest SwaI (SmiI) (ATTTAAAT)
AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA) Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC), HincII (HindII)/FastDigest HincII* MaeIII (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Alw26I (BsmAI)/FastDigest Alw26I Alw21I (BsiHKAI)/FastDigest Alw21I, Alw44I (ApaLI)/ FastDigest ApaLI (Alw44I), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest FspAI/FastDigest FspAI* (RTGCGCAC) Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) HpyCH4V FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) MaeIII, NmuCI (Tsp45I)/FastDigest NmuCI HincII (HindII)/FastDigest HincII* (GTYGAC) Hpy8I (MjaIV)/FastDigest Hpy8I MssI (PmeI)/FastDigest MssI (GTTTAAAC) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI ScaI/FastDigest ScaI (AGTACT) AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I TaqI/FastDigest TaqI Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC), HincII (HindII)/FastDigest HincII* MaeIII (GTYAAC), KspAI (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) Alw26I (BsmAI)/FastDigest Alw26I Alw21I (BsiHKAI)/FastDigest Alw21I, Alw44I (ApaLI)/ FastDigest ApaLI (Alw44I), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Hpy8I (MjaIV)/FastDigest FspAI/FastDigest FspAI* (RTGCGCAC) Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) HpyCH4V FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) MaeIII, NmuCI (Tsp45I)/FastDigest NmuCI HincII (HindII)/FastDigest HincII* (GTYGAC) Hpy8I (MjaIV)/FastDigest Hpy8I MssI (PmeI)/FastDigest MssI (GTTTAAAC) Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI RsaI/FastDigest RsaI (GTAC) DpnI/FastDigest DpnI (GATC), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/ MnlI/FastDigest MnlI FastDigest BsrBI (MbiI)* (CCGCTC) BccI Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), EheI (SfoI)/FastDigest EheI (GGCGCC), FspAI/ FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest SmuI (FauI), SsiI (AciI)/FastDigest AciI (SsiI) FspI (NsbI) (TGCGCA) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) BsaJI (BseDI), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/ FastDigest BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I SrfI (GCCCGGGC) (AvaI)/FastDigest AvaI (Eco88I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, SmaI/FastDigest SmaI AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA), HincII (HindII)/FastDigest HincII* (GTYAAC), KspAI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I (HpaI)/FastDigest HpaI (KspAI) (GTTAAC) TaqI/FastDigest TaqI Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) DraI/FastDigest DraI, FastDigest MseI (SaqAI), Tru1I DraI/FastDigest DraI (TTTAAA), MssI (PmeI)/FastDigest MssI (GTTTAAAC) (MseI)/FastDigest Tru1I FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDiHpyCH4V gest FspI (NsbI) (TGCGCA) (continued on next page)
Restriction enzymes that cleave the newly generated recognition sequence FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I TaqI/FastDigest TaqI
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SrfI (GCCCGGGC)
SspI/FastDigest SspI (AATATT)
ZraI (GACGTC)
DpnI/FastDigest DpnI (GATC), Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/ MnlI/FastDigest MnlI FastDigest BsrBI (MbiI)* (CCGCTC) BccI Eco32I (EcoRV)/FastDigest EcoRV (Eco32I) (GATATC) Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), EheI (SfoI)/FastDigest EheI (GGCGCC), FspAI/ FastDigest FspAI* (RTGCGCAC), FspAI/FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest Cac8I, SmuI (FauI), SsiI (AciI)/FastDigest AciI (SsiI) FspI (NsbI) (TGCGCA) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BseDI (BsaJI)/FastDigest MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) BsaJI (BseDI), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC) HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/ FastDigest ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/ FastDigest BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I SmaI/FastDigest SmaI (CCCGGG) (AvaI)/FastDigest AvaI (Eco88I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, SmaI/FastDigest SmaI FastDigest MseI (SaqAI), TasI (Tsp509I)/FastDigest AanI (PsiI)/FastDigest PsiI (AanI) (TTATAA) Tsp509I (TasI), Tru1I (MseI)/FastDigest Tru1I TaqI/FastDigest TaqI Bsp68I (NruI)/FastDigest NruI (RruI) (TCGCGA) Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I) (GTATAC), DpnI/FastDigest DpnI (GATC), FaiI* TasI (Tsp509I)/FastDigest Tsp509I (TasI) (YATA), FaiI* (YATG) HpyCH4V FspAI/FastDigest FspAI* (RTGCGCAC), NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) FspAI/FastDigest FspAI* (RTGCGCAT) AatII/FastDigest AatII, Hin1I (BsaHI)/FastDigest BsaHI AjiI (BmgBI)* (CACGTC) (Hin1I), MaeII, SetI, TaiI (MaeII)/FastDigest TaiI, ZraI AjiI (BmgBI)* (GACGTG), Eco105I (SnaBI)/FastDigest SnaBI (Eco105I) (TACGTA), Eco72I (PmlI)/ FastDigest PmlI (Eco72I) (CACGTG), Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTA), Ppu21I MaeII, SetI, TaiI (MaeII)/FastDigest TaiI (BsaAI)/FastDigest BsaAI (Ppu21I)* (YACGTG) AluI/FastDigest AluI (AGCT), CviJI* (RGCT), MspA1I* (CMGCTG), PvuII/FastDigest PvuII SetI (CAGCTG) HinfI/FastDigest HinfI DpnI/FastDigest DpnI (GATC) Ecl136II (EcoICRI)/FastDigest Ecl136II (GAGCTC), MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (CCGCTC) MnlI/FastDigest MnlI, SetI Eco47III (AfeI)/FastDigest AfeI (Eco47III) (AGCGCT), FspAI/FastDigest FspAI* (RTGCGCAC), FspAI/ CseI (HgaI)/FastDigest HgaI (CseI) FastDigest FspAI* (RTGCGCAT), NsbI (FspI)/FastDigest FspI (NsbI) (TGCGCA) CseI (HgaI)/FastDigest HgaI (CseI), Hin1I (BsaHI)/FastDiEheI (SfoI)/FastDigest EheI (GGCGCC) gest BsaHI (Hin1I) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI MbiI (BsrBI)/FastDigest BsrBI (MbiI)* (GAGCGG), MspA1I* (CMGCGG) BceAI PdiI (NaeI)/FastDigest NaeI (PdiI) (GCCGGC)
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Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends

Note
* Restriction enzymes that have degenerate recognition sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed. Fermentas FastDigest enzymes are shown in ruby. Fermentas conventional enzymes are shown in orange.
Single letter code

Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
First restriction enzyme AatII/FastDigest AatII (GACGTC) Second restriction enzyme SetI* (ACGT), TaiI (MaeII)/FastDigest TaiI (ACGT) Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), SmoI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG)
Restriction enzyme that cleave the newly generated recognition sequence MaeII, SetI, TaiI (MaeII)/FastDigest TaiI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), MnlI/FastDigest MnlI, SmoI (SmlI), TaqI/FastDigest TaqI, XhoI/FastDigest XhoI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), MnlI/FastDigest MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest TaqI, XhoI/FastDigest XhoI AbsI, BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), MnlI/FastDigest MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest TaqI, XhoI/FastDigest XhoI MnlI/FastDigest MnlI, TaqI/FastDigest TaqI Hpy99I, MnlI/FastDigest MnlI, TaqI/FastDigest TaqI Acc65I (Asp718I)/FastDigest Acc65I, BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), Csp6I (CviQI)/FastDigest Csp6I, KpnI/FastDigest KpnI, RsaI/FastDigest RsaI
PspXI* (VCTCGAGC), PspXI* (VCTCGAGT) AbsI (CCTCGAGG) PspXI* (VCTCGAGG) SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG) BshNI (BanI)/FastDigest BanI (BshNI)* (GGTACC) Acc65I (Asp718I)/FastDigest Acc65I (GGTACC)
AflIII* (ACATGT)
AflIII* (ACGCGT) AflIII* (ACGTGT) Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCAC) Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCTC)
Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCAC)
Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCTC) Alw44I (ApaLI)/FastDigest BfmI (SfcI)/FastDigest SfcI (BfmI)* (CTGCAG) ApaLI (Alw44I) (GTGCAC) ApaI/FastDigest ApaI (GGGCCC)
Bsp1407I (BsrGI)/FastDigest Bsp1407I (TGTACA), Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) (CGTACG), TatI/FastDigest TatI* (WGTACA), TatI/ Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI FastDigest TatI* (WGTACT) BtgI* (CCATGG), Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG), FatI (CATG), NcoI/FastDigest NcoI (CCATGG), PagI (BspHI)/FastDigest CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) BspHI (PagI) (TCATGA) AflIII, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), PscI (PciI), XceI PscI (PciI) (ACATGT) (NspI)/FastDigest NspI (XceI) BtgI* (CCGCGG) Bsh1236I (BstUI)/FastDigest Bsh1236I Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I MauBI/FastDigest MauBI (CGCGCGCG), SgsI (AscI)/FastDigest AscI (HinP1I)/FastDigest HinP1I (Hin6I) (SgsI) (GGCGCGCC), PauI (BssHII)/FastDigest BssHII (PteI) (GCGCGC) AflIII, Bsh1236I (BstUI)/FastDigest Bsh1236I, MluI/FastDigest MluI MluI/FastDigest MluI (ACGCGT) BtgI* (CCGTGG) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI Alw21I (BsiHKAI)/FastDigest Alw21I, SduI (Bsp1286I)/FastDigest SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GAGCAC) Bsp1286I (SduI) AluI/FastDigest AluI, Alw21I (BsiHKAI)/FastDigest Alw21I, CviJI, Ecl136II Eco24I (BanII)* (GAGCTC), SacI/FastDigest SacI (GAGCTC), SduI (EcoICRI)/FastDigest Ecl136II, Eco24I (BanII), SacI/FastDigest SacI, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GAGCTC) (Bsp1286I)/FastDigest Bsp1286I (SduI), SetI SetI* (AGCT) AluI/FastDigest AluI, CviJI, SetI Alw21I (BsiHKAI)/FastDigest Alw21I, Alw44I (ApaLI)/FastDigest ApaLI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GTGCAC), SduI (Alw44I), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Hpy8I (MjaIV)/ (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCAC) FastDigest Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) HpyCH4V Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) (ATGCAT) PstI/FastDigest PstI (CTGCAG), SdaI (SbfI)/FastDigest SbfI (SdaI) BsgI, HpyCH4V (CCTGCAGG) Alw21I (BsiHKAI)/FastDigest Alw21I, SduI (Bsp1286I)/FastDigest SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCTC) Bsp1286I (SduI) BsgI, HpyCH4V ApaI/FastDigest ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest Bsp120I, BspLI (NlaIV)/ FastDigest NlaIV (BspLI), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI BamHI/FastDigest BamHI, Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspLI (NlaIV)/FastDigest NlaIV (BspLI), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI BbeI, FastDigest HaeII (BfoI), BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), EheI (SfoI)/FastDigest EheI, HhaI/ FastDigest HhaI, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), Hin6I (HinP1I)/ FastDigest HinP1I (Hin6I), NarI, SspDI (KasI) FastDigest HaeII (BfoI), HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (continued on next page)
BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GGGCCC), Eco24I (BanII)* (GGGCCC), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GGGCCC)
BclI/FastDigest BclI (TGATCA), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I) (GATC), MboI/FastDigest MboI (GATC) BglII/FastDigest BglII (AGATCT), PsuI (BstYI)/FastDigest PsuI* BamHI/FastDigest BamHI (RGATCT) (GGATCC) PsuI (BstYI)/FastDigest PsuI* (RGATCC)
FastDigest HaeII (BfoI)* (RGCGCC) BbeI (GGCGCC) FastDigest HaeII (BfoI)* (RGCGCT)
www.fermentas.com
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Table 1.22.Newly generated recognition sites resulting from ligation of protruding compatible DNA ends.
Second restriction enzyme BamHI/FastDigest BamHI (GGATCC), BglII/FastDigest BglII (AGATCT), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I) (GATC), MboI/FastDigest BclI/FastDigest BclI (TGATCA) MboI (GATC), PsuI (BstYI)/FastDigest PsuI* (RGATCC), PsuI (BstYI)/FastDigest PsuI* (RGATCT) Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCTAGG), NheI/FastDigest BcuI (SpeI)/FastDigest SpeI NheI (GCTAGC), XbaI/FastDigest XbaI (TCTAGA), XmaJI (AvrII)/FastDi(BcuI) (ACTAGT) gest AvrII (XmaJI) (CCTAGG) BfmI (SfcI)/FastDigest SfcI Alw44I (ApaLI)/FastDigest ApaLI (Alw44I) (GTGCAC) (BfmI)* (CTGCAG) BamHI/FastDigest BamHI (GGATCC), PsuI (BstYI)/FastDigest PsuI* (RGATCC) BclI/FastDigest BclI (TGATCA), Bsp143I (Sau3AI)/FastDigest Sau3AI BglII/FastDigest BglII (Bsp143I) (GATC), MboI/FastDigest MboI (GATC) (AGATCT) PsuI (BstYI)/FastDigest PsuI* (RGATCT) Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC), Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC), NarI (GGCGCC), SsiI (AciI)/FastDigest AciI (SsiI)* (CCGC) HpaII/FastDigest HpaII (CCGG), MspI (HpaII)/FastDigest MspI (CCGG), SsiI (AciI)/FastDigest AciI (SsiI)* (GCGG) Bsp119I (BstBI)/FastDigest Bsp119I (TTCGAA), Bsu15I (ClaI)/FastDigest ClaI (Bsu15I) (ATCGAT), TaqI/FastDigest TaqI (TCGA), XmiI (AccI)/FastDigest AccI (XmiI)* (GTCGAC) BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), SgrAI* (CRCCGGTG) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG) Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), SgrAI* (CRCCGGTG) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG) Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GGGCAC) BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GGGCCC) SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GGGCAC) First restriction enzyme Restriction enzyme that cleave the newly generated recognition sequence Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI

FspBI (BfaI)/FastDigest BfaI (FspBI) HpyCH4V Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI BglII/FastDigest BglII, Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI SmuI (FauI), SsiI (AciI)/FastDigest AciI (SsiI) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI TaqI/FastDigest TaqI BsaWI, BshTI (AgeI)/FastDigest AgeI (BshTI), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI, HpaII/FastDigest HpaII, Hpy188III, Kpn2I (BspEI)/FastDigest Kpn2I, MspI (HpaII)/FastDigest MspI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)
BmeT110I* (CCCGRG)
BmeT110I* (CTCGRG)
BsaWI* (ACCGGW)
BsaWI* (TCCGGW)
BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GTGCAC) BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GTGCCC) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)* (CGATCG) BshNI (BanI)/FastDigest BanI (BshNI)* (GGCGCC)
ApaI/FastDigest ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest Bsp120I, BspLI (NlaIV)/ ApaI/FastDigest ApaI (GGGCCC), Eco24I (BanII)* (GGGCCC), SduI FastDigest NlaIV (BspLI), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GGGCCC) (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI) Alw21I (BsiHKAI)/FastDigest Alw21I, Alw44I (ApaLI)/FastDigest ApaLI Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCAC), SduI (Bsp1286I)/FastDi(Alw44I), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Hpy8I (MjaIV)/ gest Bsp1286I (SduI)* (GTGCAC) FastDigest Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) HpyCH4V Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) (ATGCAT) PstI/FastDigest PstI (CTGCAG), SdaI (SbfI)/FastDigest SbfI (SdaI) BsgI, HpyCH4V (CCTGCAGG)
SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCCC) PacI/FastDigest PacI (TTAATTAA) PvuI/FastDigest PvuI (CGATCG), SfaAI (AsiSI)/FastDigest AsiSI (SfaAI) (GCGATCGC)

BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI BbeI, FastDigest HaeII (BfoI), BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), EheI (SfoI)/FastDigest EheI, HhaI/ FastDigest HhaI, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), Hin6I (HinP1I)/ FastDigest HinP1I (Hin6I), NarI, SspDI (KasI) Acc65I (Asp718I)/FastDigest Acc65I, BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), Csp6I (CviQI)/FastDigest Csp6I, KpnI/FastDigest KpnI, RsaI/FastDigest RsaI
SspDI (KasI) (GGCGCC)
Acc65I (Asp718I)/FastDigest Acc65I (GGTACC) BshNI (BanI)/FastDigest BanI (BshNI)* (GGTACC)
Bsp1407I (BsrGI)/FastDigest Bsp1407I (TGTACA), Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) (CGTACG), TatI/FastDigest TatI* (WGTACA), TatI/ Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI FastDigest TatI* (WGTACT) (continued on next page)
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BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT)
Bsp119I (BstBI)/FastDigest Bsp119I (TTCGAA)
Bsp120I (PspOMI)/FastDigest Bsp120I (GGGCCC)
Second restriction enzyme BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BsaWI* (WCCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), SgrAI* (CRCCGGTG) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG) Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) BmeT110I* (CYCGAG), Bsu15I (ClaI)/FastDigest ClaI (Bsu15I) (ATCGAT), TaqI/FastDigest TaqI (TCGA), XmiI (AccI)/FastDigest AccI (XmiI)* (GTCGAC) CfrI (EaeI)* (YGGCCA), CfrI (EaeI)* (YGGCCG), Eco52I (EagI)/FastDigest EagI (Eco52I) (CGGCCG) NotI/FastDigest NotI (GCGGCCGC) Acc65I (Asp718I)/FastDigest Acc65I (GGTACC), BshNI (BanI)/FastDigest BanI (BshNI)* (GGTACC), Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) (CGTACG)
Restriction enzyme that cleave the newly generated recognition sequence BsaWI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI, BshTI (AgeI)/FastDigest AgeI (BshTI), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI TaqI/FastDigest TaqI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDigest AciI (SsiI), TauI/FastDigest TauI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bsp1407I (BsrGI)/FastDigest Bsp1407I, Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI, TatI/FastDigest TatI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI, TatI/FastDigest TatI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI BspTI (AflII)/FastDigest AflII (BspTI), FastDigest MseI (SaqAI), SmoI (SmlI), Tru1I (MseI)/FastDigest Tru1I TaqI/FastDigest TaqI MaeII, SetI, TaiI (MaeII)/FastDigest TaiI CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) BseDI (BsaJI)/FastDigest BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDigest StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), NcoI/ FastDigest NcoI Bsh1236I (BstUI)/FastDigest Bsh1236I, SsiI (AciI)/FastDigest AciI (SsiI) Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I), SsiI (AciI)/FastDigest AciI (SsiI) BsaWI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI, BshTI (AgeI)/FastDigest AgeI (BshTI), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI) SsiI (AciI)/FastDigest AciI (SsiI)
Bsp1407I (BsrGI)/FastDigest Bsp1407I (TGTACA) TatI/FastDigest TatI* (WGTACA) TatI/FastDigest TatI* (WGTACT) Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I) (GATC) BspTI (AflII)/FastDigest AflII (BspTI) (CTTAAG) Bsu15I (ClaI)/FastDigest ClaI (Bsu15I) (ATCGAT) BtgI* (CCACGG)
BamHI/FastDigest BamHI (GGATCC), BclI/FastDigest BclI (TGATCA), BglII/FastDigest BglII (AGATCT), MboI/FastDigest MboI (GATC), PsuI (BstYI)/FastDigest PsuI* (RGATCC), PsuI (BstYI)/FastDigest PsuI* (RGATCT) SmoI (SmlI)* (CTTAAG) BmeT110I* (CYCGAG), Bsp119I (BstBI)/FastDigest Bsp119I (TTCGAA), TaqI/FastDigest TaqI (TCGA), XmiI (AccI)/FastDigest AccI (XmiI)* (GTCGAC) AflIII* (ACACGT) AflIII* (ACATGT), FatI (CATG), PagI (BspHI)/FastDigest BspHI (PagI) (TCATGA), PscI (PciI) (ACATGT) Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG), NcoI/FastDigest NcoI (CCATGG)
BtgI* (CCATGG)
BtgI* (CCGCGG)
Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (ACCGGY)
Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (GCCGGY)
Cfr42I (SacII) (CCGCGG)
Cfr9I (XmaI) (CCCGGG)
AflIII* (ACGCGT), MluI/FastDigest MluI (ACGCGT) MauBI/FastDigest MauBI (CGCGCGCG), SgsI (AscI)/FastDigest AscI (SgsI) (GGCGCGCC), PauI (BssHII)/FastDigest BssHII (PteI) (GCGCGC) BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), SgrAI* (CRCCGGTG) Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG) BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), SgrAI* (CRCCGGTG) Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)* (CGGCCG) BsaWI* (WCCGGA), BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), Kpn2I (BspEI)/FastDi(Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI gest Kpn2I (TCCGGA), MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG), SgrAI* (CRCCGGTG) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest BsaJI (BseDI), BssKI, Cfr9I Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) (XmaI), Eco88I (AvaI)/FastDigest AvaI (Eco88I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, SmaI/FastDigest SmaI (continued on next page)
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First restriction enzyme Restriction enzyme that cleave the newly generated recognition sequence BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDiBsp120I (PspOMI)/FastDigest Bsp120I (GGGCCC) gest Sau96I (Cfr13I), CviJI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest Eco52I (EagI)/FastDigest EagI (Eco52I) (CGGCCG) HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), SatI NotI/FastDigest NotI (GCGGCCGC) (Fnu4HI)/FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDigest AciI (SsiI), TauI/ FastDigest TauI BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDi Bsp120I (PspOMI)/FastDigest Bsp120I (GGGCCC) gest Sau96I (Cfr13I), CviJI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI Eco52I (EagI)/FastDigest EagI (Eco52I) (CGGCCG) BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, SatI (Fnu4HI)/ FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDigest AciI (SsiI), TauI/FastDiNotI/FastDigest NotI (GCGGCCGC) gest TauI BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGACC) FastDigest AvaII (Eco47I) FastDigest SanDI (KflI)* (GGGACCC), Psp5II (PpuMI)/FastDigest PpuMI BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDi(Psp5II)* (RGGACCC) gest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I) BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGACCT) FastDigest AvaII (Eco47I), SetI Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGTCC), Psp5II (PpuMI)/FastDi- BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ gest PpuMI (Psp5II)* (RGGTCCT) FastDigest AvaII (Eco47I) FastDigest SanDI (KflI)* (GGGTCCC), Psp5II (PpuMI)/FastDigest PpuMI BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I) (Psp5II)* (RGGTCCC) NdeI/FastDigest NdeI (CATATG) XmiI (AccI)/FastDigest AccI (XmiI)* (GTATAC) AflIII* (ACATGT), FatI (CATG), PagI (BspHI)/FastDigest BspHI (PagI) (TCATGA), PscI (PciI) (ACATGT) BtgI* (CCATGG), NcoI/FastDigest NcoI (CCATGG) BcuI (SpeI)/FastDigest SpeI (BcuI) (ACTAGT), NheI/FastDigest NheI (GCTAGC), XbaI/FastDigest XbaI (TCTAGA) XmaJI (AvrII)/FastDigest AvrII (XmaJI) (CCTAGG) SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GAGCCC) Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCTC), SacI/FastDigest SacI (GAGCTC), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GAGCTC) SetI* (AGCT) ApaI/FastDigest ApaI (GGGCCC), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GGGCCC), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GGGCCC) SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GGGCTC) CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGACCG) Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGACC)
CfrI (EaeI)* (CGGCCR)
CfrI (EaeI)* (TGGCCR)
CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGACCG)
CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGTCCG) Csp6I (CviQI)/FastDigest Csp6I (GTAC) CviAII (CATG) Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG)
FaiI FaiI CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) BseDI (BsaJI)/FastDigest BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDigest StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), NcoI/ FastDigest NcoI FspBI (BfaI)/FastDigest BfaI (FspBI) BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI (Eco130I), FspBI (BfaI)/FastDigest BfaI (FspBI), XmaJI (AvrII)/FastDigest AvrII (XmaJI) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) AluI/FastDigest AluI, Alw21I (BsiHKAI)/FastDigest Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest Ecl136II, Eco24I (BanII), SacI/FastDigest SacI, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI), SetI AluI/FastDigest AluI, CviJI, SetI ApaI/FastDigest ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest Bsp120I, BspLI (NlaIV)/ FastDigest NlaIV (BspLI), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ FastDigest AvaII (Eco47I) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I) BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ FastDigest AvaII (Eco47I), SetI BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ FastDigest AvaII (Eco47I) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I) BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDigest AciI (SsiI), TauI/ FastDigest TauI (continued on next page)
Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCTAGG)
Eco24I (BanII)* (GAGCCC) Eco24I (BanII)* (GAGCTC)
Eco24I (BanII)* (GGGCCC)
Eco24I (BanII)* (GGGCTC)
FastDigest SanDI (KflI)* (GGGACCC), Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGACCC) Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGACCT)
Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGTCC)
CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGTCCG), Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGTCCT) FastDigest SanDI (KflI)* (GGGTCCC), Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGTCCC) Bsp120I (PspOMI)/FastDigest Bsp120I (GGGCCC) CfrI (EaeI)* (YGGCCA)
Eco52I (EagI)/FastDigest EagI (Eco52I) (CGGCCG)
CfrI (EaeI)* (YGGCCG)
NotI/FastDigest NotI (GCGGCCGC)
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Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG)
Second restriction enzyme BsaWI* (WCCGGA), BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA), MreI (Sse232I)/FastDigest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG), SgrAI* (CRCCGGTG) Cfr9I (XmaI) (CCCGGG)
Restriction enzyme that cleave the newly generated recognition sequence BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BmeT110I, BseDI (BsaJI)/FastDigest BsaJI (BseDI), BssKI, Cfr9I (XmaI), Eco88I (AvaI)/FastDigest AvaI (Eco88I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, SmaI/FastDigest SmaI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), SmoI (SmlI), TaqI/ FastDigest TaqI, XhoI/FastDigest XhoI TaqI/FastDigest TaqI Hpy99I, TaqI/FastDigest TaqI
Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG)
EcoRI/FastDigest EcoRI (GAATTC)
EcoRII* (CCAGG) EcoRII* (CCTGG) FatI (CATG) FspBI (BfaI)/FastDigest BfaI (FspBI) (CTAG) FastDigest HaeII (BfoI)* (AGCGCY) FastDigest HaeII (BfoI)* (GGCGCY)
AbsI (CCTCGAGG), PspXI* (VCTCGAGC), PspXI* (VCTCGAGG), PspXI* (VCTCGAGT), SmoI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG) SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG) MunI (MfeI)/FastDigest MfeI (MunI) (CAATTG), TasI (Tsp509I)/FastDigest TasI (Tsp509I)/FastDigest Tsp509I (TasI) Tsp509I (TasI) (AATT) EcoRI/FastDigest EcoRI, TasI (Tsp509I)/FastDigest Tsp509I (TasI), XapI XapI (ApoI)/FastDigest XapI* (RAATTC) (ApoI)/FastDigest XapI TasI (Tsp509I)/FastDigest Tsp509I (TasI), XapI (ApoI)/FastDigest XapI XapI (ApoI)/FastDigest XapI* (RAATTT) Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/ FastDigest SexAI (CsiI)* (ACCAGGT) FastDigest MvaI, SetI Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/ FastDigest SexAI (CsiI)* (ACCTGGT) FastDigest MvaI AflIII* (ACATGT), BtgI* (CCATGG), Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG), NcoI/FastDigest NcoI (CCATGG), PagI (BspHI)/FastDigest CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) BspHI (PagI) (TCATGA), PscI (PciI) (ACATGT) NdeI/FastDigest NdeI (CATATG) BbeI (GGCGCC) FaiI FastDigest HaeII (BfoI), HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) BbeI, FastDigest HaeII (BfoI), BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), EheI (SfoI)/FastDigest EheI, HhaI/ FastDigest HhaI, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), Hin6I (HinP1I)/ FastDigest HinP1I (Hin6I), NarI, SspDI (KasI) CseI (HgaI)/FastDigest HgaI (CseI) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI CseI (HgaI)/FastDigest HgaI (CseI), Hin1I (BsaHI)/FastDigest BsaHI (Hin1I) HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) BbeI, FastDigest HaeII (BfoI), BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), EheI (SfoI)/FastDigest EheI, HhaI/ FastDigest HhaI, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), Hin6I (HinP1I)/ FastDigest HinP1I (Hin6I), NarI, SspDI (KasI) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI
BbeI (GGCGCC) Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC), SsiI (AciI)/FastDigest AciI (SsiI)* (CCGC) MaeII (ACGT), Psp1406I (AclI)/FastDigest AclI (Psp1406I) (AACGTT) NarI (GGCGCC) Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC), SsiI (AciI)/FastDigest AciI (SsiI)* (CCGC) NarI (GGCGCC) PaeI (SphI)/FastDigest SphI (PaeI) (GCATGC), XceI (NspI)/FastDigest NspI (XceI)* (RCATGC), XceI (NspI)/FastDigest NspI (XceI)* (RCATGT) Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC), NarI (GGCGCC), SsiI (AciI)/FastDigest AciI (SsiI)* (CCGC) BmeT110I* (CYCGGG)
Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GACGYC)
Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GGCGYC)
Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) (CATG) Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC)
Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC), Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC), NarI (GGCGCC), SsiI (AciI)/FastDigest AciI SsiI (AciI)/FastDigest AciI (SsiI) (SsiI)* (CCGC) MspI (HpaII)/FastDigest MspI (CCGG), SsiI (AciI)/FastDigest AciI (SsiI)* HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI (GCGG) BsaWI, HpaII/FastDigest HpaII, Hpy188III, Kpn2I (BspEI)/FastDigest BsaWI* (WCCGGA) Kpn2I, MspI (HpaII)/FastDigest MspI BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), BsaWI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Kpn2I (BspEI)/FastDigest Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), SgrAI* (CRCCGGTG) Kpn2I (TCCGGA) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), MreI (Sse232I)/FastHpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Digest MreI (CGCCGGCG), NgoMIV (GCCGGC), SgrAI* (CRCCGGCG) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGTC), Psp1406I (AclI)/FastDiMaeII, SetI, TaiI (MaeII)/FastDigest TaiI MaeII (ACGT) gest AclI (Psp1406I) (AACGTT) Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I AflIII* (ACGCGT), BtgI* (CCGCGG), MluI/FastDigest MluI (ACGCGT) (HinP1I)/FastDigest HinP1I (Hin6I) MauBI/FastDigest MauBI (CGCGCGCG) PauI (BssHII)/FastDigest BssHII (PteI) (GCGCGC), SgsI (AscI)/FastDigest Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I AscI (SgsI) (GGCGCGCC) (HinP1I)/FastDigest HinP1I (Hin6I), PauI (BssHII)/FastDigest BssHII (PteI) HpaII/FastDigest HpaII (CCGG) (continued on next page)
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Second restriction enzyme BamHI/FastDigest BamHI (GGATCC), BclI/FastDigest BclI (TGATCA), BglII/FastDigest BglII (AGATCT), Bsp143I (Sau3AI)/FastDigest Sau3AI MboI/FastDigest MboI (GATC) (Bsp143I) (GATC), PsuI (BstYI)/FastDigest PsuI* (RGATCC), PsuI (BstYI)/ FastDigest PsuI* (RGATCT) AflIII* (ACGCGT) BtgI* (CCGCGG) MluI/FastDigest MluI (ACGCGT) MauBI/FastDigest MauBI (CGCGCGCG), SgsI (AscI)/FastDigest AscI (SgsI) (GGCGCGCC), PauI (BssHII)/FastDigest BssHII (PteI) (GCGCGC) Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCAC), BseSI (Bme1580I)/FastMph1103I (NsiI)/FastDigest Digest Bme1580I (BseSI)* (GTGCAC), PstI/FastDigest PstI (CTGCAG), NsiI (Mph1103I) (ATGCAT) SdaI (SbfI)/FastDigest SbfI (SdaI) (CCTGCAGG), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCAC) BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), NgoMIV (GCCGGC) MreI (Sse232I)/FastDigest Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* MreI (CGCCGGCG) (CCCGGG) SgrAI* (CRCCGGCG) SgrAI* (CRCCGGTG) FastDigest MseI (SaqAI) (TTAA) First restriction enzyme Restriction enzyme that cleave the newly generated recognition sequence Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI

AflIII, Bsh1236I (BstUI)/FastDigest Bsh1236I, MluI/FastDigest MluI Bsh1236I (BstUI)/FastDigest Bsh1236I Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) HpyCH4V HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MreI (Sse232I)/FastDigest MreI, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI), SgrAI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, SgrAI FaiI
MspI (HpaII)/FastDigest MspI (CCGG)
MunI (MfeI)/FastDigest MfeI (MunI) (CAATTG)
NarI (GGCGCC)
NcoI/FastDigest NcoI (CCATGG)
NdeI/FastDigest NdeI (CATATG)
NgoMIV (GCCGGC)
NheI/FastDigest NheI (GCTAGC)
NotI/FastDigest NotI (GCGGCCGC)
NdeI/FastDigest NdeI (CATATG) Tru1I (MseI)/FastDigest Tru1I (TTAA), VspI (AseI)/FastDigest AseI (VspI) FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I (ATTAAT) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI BmeT110I* (CYCGGG) (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC), Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC), NarI (GGCGCC), SsiI (AciI)/FastDigest AciI SsiI (AciI)/FastDigest AciI (SsiI) (SsiI)* (CCGC) HpaII/FastDigest HpaII (CCGG), SsiI (AciI)/FastDigest AciI (SsiI)* HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI (GCGG) EcoRI/FastDigest EcoRI (GAATTC), TasI (Tsp509I)/FastDigest Tsp509I (TasI) (AATT), XapI (ApoI)/FastDigest XapI* (RAATTC), XapI (ApoI)/FastDi- TasI (Tsp509I)/FastDigest Tsp509I (TasI) gest XapI* (RAATTT) BbeI, FastDigest HaeII (BfoI), BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), EheI (SfoI)/FastDigest EheI, HhaI/ Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC) FastDigest HhaI, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), Hin6I (HinP1I)/ FastDigest HinP1I (Hin6I), NarI, SspDI (KasI) Hin1I (BsaHI)/FastDigest BsaHI (Hin1I) Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGTC) Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) (GCGC), SsiI (AciI)/FastDigest HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) AciI (SsiI)* (CCGC) AflIII* (ACATGT), FatI (CATG), PagI (BspHI)/FastDigest BspHI (PagI) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) (TCATGA), PscI (PciI) (ACATGT) BseDI (BsaJI)/FastDigest BsaJI (BseDI), BtgI, CviAII, Eco130I (StyI)/FastDigest StyI (Eco130I), FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), NcoI/ BtgI* (CCATGG), Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG) FastDigest NcoI Csp6I (CviQI)/FastDigest Csp6I (GTAC), FspBI (BfaI)/FastDigest BfaI (FspBI) (CTAG), FastDigest MseI (SaqAI) (TTAA), Tru1I (MseI)/FastDigest FaiI Tru1I (TTAA), VspI (AseI)/FastDigest AseI (VspI) (ATTAAT) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT), SgrAI* (CRCCGGTG) (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), MreI (Sse232I)/Fast- Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, Digest MreI (CGCCGGCG), SgrAI* (CRCCGGCG) MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BcuI (SpeI)/FastDigest SpeI (BcuI) (ACTAGT), Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCTAGG), XbaI/FastDigest XbaI (TCTAGA), XmaJI (AvrII)/ FspBI (BfaI)/FastDigest BfaI (FspBI) FastDigest AvrII (XmaJI) (CCTAGG) BmgT120I, BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDiBsp120I (PspOMI)/FastDigest Bsp120I (GGGCCC) gest Sau96I (Cfr13I), CviJI, SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/ FastDigest TauI BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, SatI (Fnu4HI)/ CfrI (EaeI)* (YGGCCA) FastDigest Fnu4HI (SatI), TauI/FastDigest TauI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest CfrI (EaeI)* (YGGCCG), Eco52I (EagI)/FastDigest EagI (Eco52I) (CGGCCG) HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/FastDigest TauI (continued on next page)
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First restriction enzyme PacI/FastDigest PacI (TTAATTAA)
Second restriction enzyme Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)* (CGATCG), PvuI/FastDigest PvuI (CGATCG), SfaAI (AsiSI)/FastDigest AsiSI (SfaAI) (GCGATCGC) Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) (CATG)
Restriction enzyme that cleave the newly generated recognition sequence FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), PaeI (SphI)/ FastDigest SphI (PaeI), XceI (NspI)/FastDigest NspI (XceI) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), XceI (NspI)/FastDigest NspI (XceI)
PaeI (SphI)/FastDigest SphI XceI (NspI)/FastDigest NspI (XceI)* (RCATGC) (PaeI) (GCATGC) XceI (NspI)/FastDigest NspI (XceI)* (RCATGT) PagI (BspHI)/FastDigest BspHI (PagI) (TCATGA) PasI* (CCCAGGG) PauI (BssHII)/FastDigest BssHII (PteI) (GCGCGC)
Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) (CGTACG)
PscI (PciI) (ACATGT)
AflIII* (ACATGT), BtgI* (CCATGG), Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG), FatI (CATG), NcoI/FastDigest NcoI (CCATGG), PscI (PciI) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) (ACATGT) TseI* (GCAGC) BseYI Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I AflIII* (ACGCGT), BtgI* (CCGCGG), MluI/FastDigest MluI (ACGCGT) (HinP1I)/FastDigest HinP1I (Hin6I) Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I MauBI/FastDigest MauBI (CGCGCGCG), SgsI (AscI)/FastDigest AscI (SgsI) (GGCGCGCC) (HinP1I)/FastDigest HinP1I (Hin6I), PauI (BssHII)/FastDigest BssHII (PteI) Acc65I (Asp718I)/FastDigest Acc65I (GGTACC), BshNI (BanI)/FastDigest BanI (BshNI)* (GGTACC), Bsp1407I (BsrGI)/FastDigest Bsp1407I Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI (TGTACA), TatI/FastDigest TatI* (WGTACA), TatI/FastDigest TatI* (WGTACT) AflIII, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), PscI (PciI), XceI AflIII* (ACATGT) (NspI)/FastDigest NspI (XceI) BtgI* (CCATGG), Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCATGG), FatI (CATG), NcoI/FastDigest NcoI (CCATGG), PagI (BspHI)/FastDigest CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) BspHI (PagI) (TCATGA) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ FastDigest AvaII (Eco47I) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, Psp5II (PpuMI)/FastDigest PpuMI (Psp5II) BmgT120I, Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/ FastDigest AvaII (Eco47I), SetI BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, Psp5II (PpuMI)/FastDigest PpuMI (Psp5II), SetI BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), FaqI (BsmFI)/FastDigest BsmFI (FaqI) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, FaqI (BsmFI)/FastDigest BsmFI (FaqI), FastDigest SanDI (KflI), Psp5II (PpuMI)/FastDigest PpuMI (Psp5II) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, FastDigest SanDI (KflI), Psp5II (PpuMI)/ FastDigest PpuMI (Psp5II) BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), PspXI, SmoI (SmlI), TaqI/ FastDigest TaqI, XhoI/FastDigest XhoI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), SmoI (SmlI), TaqI/ FastDigest TaqI, XhoI/FastDigest XhoI TaqI/FastDigest TaqI Hpy99I, TaqI/FastDigest TaqI AbsI, BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), MnlI/FastDigest MnlI, PspXI, SmoI (SmlI), TaqI/FastDigest TaqI, XhoI/FastDigest XhoI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), MnlI/FastDigest MnlI, SmoI (SmlI), TaqI/FastDigest TaqI, XhoI/FastDigest XhoI MnlI/FastDigest MnlI, TaqI/FastDigest TaqI Hpy99I, MnlI/FastDigest MnlI, TaqI/FastDigest TaqI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), PspXI, SmoI (SmlI), TaqI/ FastDigest TaqI, XhoI/FastDigest XhoI BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), SmoI (SmlI), TaqI/ FastDigest TaqI, XhoI/FastDigest XhoI TaqI/FastDigest TaqI Hpy99I, TaqI/FastDigest TaqI (continued on next page)
Psp1406I (AclI)/FastDigest Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGTC), MaeII (ACGT) AclI (Psp1406I) (AACGTT) CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGACCG), Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGACC) Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (AGGACCY) FastDigest SanDI (KflI)* (GGGACCC) CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGTCCG), Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGTCC) Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (AGGTCCY) FastDigest SanDI (KflI)* (GGGTCCC)
CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGACCG), Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGACC) Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (GGGACCY) FastDigest SanDI (KflI)* (GGGACCC) CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGTCCG), Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGTCC) Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (GGGTCCY) FastDigest SanDI (KflI)* (GGGTCCC)
AbsI (CCTCGAGG) PspXI* (ACTCGAGB) Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), SmoI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG) SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG) AbsI (CCTCGAGG) PspXI* (CCTCGAGB) Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), SmoI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG) SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG) AbsI (CCTCGAGG) PspXI* (GCTCGAGB) Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), SmoI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG) SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG)
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First restriction enzyme PstI/FastDigest PstI (CTGCAG) Second restriction enzyme Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCAC), BseSI (Bme1580I)/ FastDigest Bme1580I (BseSI)* (GTGCAC), Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) (ATGCAT), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCAC) SdaI (SbfI)/FastDigest SbfI (SdaI) (CCTGCAGG) BamHI/FastDigest BamHI (GGATCC)
Restriction enzyme that cleave the newly generated recognition sequence HpyCH4V BfmI (SfcI)/FastDigest SfcI (BfmI), HpyCH4V, PstI/FastDigest PstI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI BglII/FastDigest BglII, Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI BamHI/FastDigest BamHI, Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspLI (NlaIV)/FastDigest NlaIV (BspLI), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), BspPI (AlwI), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI (BstYI)/FastDigest PsuI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I AluI/FastDigest AluI, Alw21I (BsiHKAI)/FastDigest Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest Ecl136II, Eco24I (BanII), SacI/FastDigest SacI, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI), SetI AluI/FastDigest AluI, CviJI, SetI TaqI/FastDigest TaqI HincII (HindII)/FastDigest HincII, Hpy8I (MjaIV)/FastDigest Hpy8I, Hpy99I, SalI/FastDigest SalI, TaqI/FastDigest TaqI, XmiI (AccI)/FastDigest AccI (XmiI) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), FaqI (BsmFI)/FastDigest BsmFI (FaqI) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, FaqI (BsmFI)/FastDigest BsmFI (FaqI), FastDigest SanDI (KflI), Psp5II (PpuMI)/FastDigest PpuMI (Psp5II) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, FaqI (BsmFI)/FastDigest BsmFI (FaqI), Psp5II (PpuMI)/FastDigest PpuMI (Psp5II), SetI BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, FastDigest SanDI (KflI), Psp5II (PpuMI)/ FastDigest PpuMI (Psp5II) BmgT120I, BspLI (NlaIV)/FastDigest NlaIV (BspLI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), Eco47I (AvaII)/FastDigest AvaII (Eco47I), EcoO109I (DraII)/FastDigest EcoO109I, Psp5II (PpuMI)/FastDigest PpuMI (Psp5II) HpyCH4V BfmI (SfcI)/FastDigest SfcI (BfmI), HpyCH4V, PstI/FastDigest PstI Alw21I (BsiHKAI)/FastDigest Alw21I, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) AluI/FastDigest AluI, Alw21I (BsiHKAI)/FastDigest Alw21I, CviJI, Ecl136II (EcoICRI)/FastDigest Ecl136II, Eco24I (BanII), SacI/FastDigest SacI, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI), SetI AluI/FastDigest AluI, CviJI, SetI BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) (continued on next page)
PsuI (BstYI)/FastDigest PsuI* (AGATCY)
BclI/FastDigest BclI (TGATCA), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I) (GATC), MboI/FastDigest MboI (GATC) BglII/FastDigest BglII (AGATCT)
BamHI/FastDigest BamHI (GGATCC) PsuI (BstYI)/FastDigest PsuI* (GGATCY)
BclI/FastDigest BclI (TGATCA), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I) (GATC), MboI/FastDigest MboI (GATC) BglII/FastDigest BglII (AGATCT)
PvuI/FastDigest PvuI (CGATCG)
Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)* (CGATCG), SfaAI (AsiSI)/ FastDigest AsiSI (SfaAI) (GCGATCGC) PacI/FastDigest PacI (TTAATTAA)
SacI/FastDigest SacI (GAGCTC)
Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCTC), Eco24I (BanII)* (GAGCTC), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GAGCTC) SetI* (AGCT) AbsI (CCTCGAGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), PspXI* (VCTCGAGC), PspXI* (VCTCGAGG), PspXI* (VCTCGAGT), SmoI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG) SgrDI (CGTCGACG) CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGACCG), Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGACC)
SalI/FastDigest SalI (GTCGAC)
FastDigest SanDI (KflI)* (GGGACCC)
Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGACCC)
Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGACCT) CpoI (RsrII)/FastDigest RsrII (CpoI)* (CGGTCCG), Eco47I (AvaII)/FastDigest AvaII (Eco47I)* (GGTCC) FastDigest SanDI (KflI)* (GGGTCCC) Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGTCCC)
Psp5II (PpuMI)/FastDigest PpuMI (Psp5II)* (RGGTCCT) Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCAC), BseSI (Bme1580I)/ FastDigest Bme1580I (BseSI)* (GTGCAC), Mph1103I (NsiI)/FastDigest SdaI (SbfI)/FastDigest SbfI NsiI (Mph1103I) (ATGCAT), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (SdaI) (CCTGCAGG) (GTGCAC) PstI/FastDigest PstI (CTGCAG) SduI (Bsp1286I)/FastDigest Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCAC) Bsp1286I (SduI)* (GAGCAC) SduI (Bsp1286I)/FastDigest Eco24I (BanII)* (GAGCCC) Bsp1286I (SduI)* (GAGCCC)
Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCTC), Eco24I (BanII)* SduI (Bsp1286I)/FastDigest (GAGCTC), SacI/FastDigest SacI (GAGCTC) Bsp1286I (SduI)* (GAGCTC) SetI* (AGCT) SduI (Bsp1286I)/FastDigest BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GGGCAC) Bsp1286I (SduI)* (GGGCAC)
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SduI (Bsp1286I)/FastDigest ApaI/FastDigest ApaI (GGGCCC), BseSI (Bme1580I)/FastDigest Bsp1286I (SduI)* (GGGCCC) Bme1580I (BseSI)* (GGGCCC), Eco24I (BanII)* (GGGCCC) SduI (Bsp1286I)/FastDigest Eco24I (BanII)* (GGGCTC) Bsp1286I (SduI)* (GGGCTC)
Restriction enzyme that cleave the newly generated recognition sequence ApaI/FastDigest ApaI, BmgT120I, BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Bsp120I (PspOMI)/FastDigest Bsp120I, BspLI (NlaIV)/ FastDigest NlaIV (BspLI), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, Eco24I (BanII), SduI (Bsp1286I)/ FastDigest Bsp1286I (SduI) CviJI, Eco24I (BanII), SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)
SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCAC)
SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCCC) SduI (Bsp1286I)/FastDigest Bsp1286I (SduI)* (GTGCTC) SetI* (ACGT) SetI* (AGCT) FastDigest SexAI (CsiI)* (ACCAGGT) FastDigest SexAI (CsiI)* (ACCTGGT) SfaAI (AsiSI)/FastDigest AsiSI (SfaAI) (GCGATCGC)
SgrAI* (CACCGGYG)
SgrAI* (CGCCGGYG)
SgrDI (CGTCGACG)
SgsI (AscI)/FastDigest AscI (SgsI) (GGCGCGCC)
SmoI (SmlI)* (CTCGAG)
SmoI (SmlI)* (CTTAAG)
SsiI (AciI)/FastDigest AciI (SsiI)* (CCGC)
Alw21I (BsiHKAI)/FastDigest Alw21I, Alw44I (ApaLI)/FastDigest ApaLI Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCAC), BseSI (Bme1580I)/Fast(Alw44I), BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), Hpy8I (MjaIV)/ Digest Bme1580I (BseSI)* (GTGCAC) FastDigest Hpy8I, HpyCH4V, SduI (Bsp1286I)/FastDigest Bsp1286I (SduI) HpyCH4V Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) (ATGCAT) PstI/FastDigest PstI (CTGCAG), SdaI (SbfI)/FastDigest SbfI (SdaI) BsgI, HpyCH4V (CCTGCAGG) BseSI (Bme1580I)/FastDigest Bme1580I (BseSI), SduI (Bsp1286I)/FastDi BseSI (Bme1580I)/FastDigest Bme1580I (BseSI)* (GTGCCC) gest Bsp1286I (SduI) Alw21I (BsiHKAI)/FastDigest Alw21I, SduI (Bsp1286I)/FastDigest Alw21I (BsiHKAI)/FastDigest Alw21I* (GTGCTC) Bsp1286I (SduI) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI AatII/FastDigest AatII (GACGTC), TaiI (MaeII)/FastDigest TaiI (ACGT) Alw21I (BsiHKAI)/FastDigest Alw21I* (GAGCTC), Eco24I (BanII)* (GAGCTC), SacI/FastDigest SacI (GAGCTC), SduI (Bsp1286I)/FastDiAluI/FastDigest AluI, CviJI, SetI gest Bsp1286I (SduI)* (GAGCTC) Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/ EcoRII* (CCAGG) FastDigest MvaI Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I), BssKI, EcoRII, MvaI (BstNI)/ EcoRII* (CCTGG) FastDigest MvaI, SetI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDi Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)* (CGATCG), PvuI/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, gest PvuI (CGATCG) PvuI/FastDigest PvuI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I PacI/FastDigest PacI (TTAATTAA) BsaWI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) BsaWI, BshTI (AgeI)/FastDigest AgeI (BshTI), Cfr10I (BsrFI)/FastDigest BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT) BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), NgoMIV (GCCGGC) (HpaII)/FastDigest MspI BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI MreI (Sse232I)/FastDigest MreI (CGCCGGCG) (HpaII)/FastDigest MspI, SgrAI HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BsaWI* (WCCGGA), Kpn2I (BspEI)/FastDigest Kpn2I (TCCGGA) Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MspI BsaWI* (WCCGGT), BshTI (AgeI)/FastDigest AgeI (BshTI) (ACCGGT), Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGT) (HpaII)/FastDigest MspI Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)* (RCCGGC), NgoMIV (GCCGGC) MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI) BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI Cfr9I (XmaI) (CCCGGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CCCGGG) (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), HpaII/FastDigest HpaII, MreI (Sse232I)/FastDigest MreI, MspI (HpaII)/FastDigest MspI, NgoMIV, MreI (Sse232I)/FastDigest MreI (CGCCGGCG) PdiI (NaeI)/FastDigest NaeI (PdiI), SgrAI AbsI (CCTCGAGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), PspXI* (VCTCGAGC), PspXI* (VCTCGAGG), PspXI* (VCTCGAGT), SmoI Hpy99I, TaqI/FastDigest TaqI (SmlI)* (CTCGAG), XhoI/FastDigest XhoI (CTCGAG) HincII (HindII)/FastDigest HincII, Hpy8I (MjaIV)/FastDigest Hpy8I, Hpy99I, SalI/FastDigest SalI, TaqI/FastDigest TaqI, XmiI (AccI)/FastDigest AccI SalI/FastDigest SalI (GTCGAC) (XmiI) Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I AflIII* (ACGCGT), BtgI* (CCGCGG), MluI/FastDigest MluI (ACGCGT) (HinP1I)/FastDigest HinP1I (Hin6I) MauBI/FastDigest MauBI (CGCGCGCG), PauI (BssHII)/FastDigest BssHII Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I (PteI) (GCGCGC) (HinP1I)/FastDigest HinP1I (Hin6I), PauI (BssHII)/FastDigest BssHII (PteI) AbsI (CCTCGAGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), SmoI (SmlI), TaqI/ PspXI* (VCTCGAGC), PspXI* (VCTCGAGG), PspXI* (VCTCGAGT), XhoI/ FastDigest TaqI, XhoI/FastDigest XhoI FastDigest XhoI (CTCGAG) TaqI/FastDigest TaqI SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG) Hpy99I, TaqI/FastDigest TaqI BspTI (AflII)/FastDigest AflII (BspTI), FastDigest MseI (SaqAI), SmoI (SmlI), BspTI (AflII)/FastDigest AflII (BspTI) (CTTAAG) Tru1I (MseI)/FastDigest Tru1I BcnI (NciI)/FastDigest NciI (BcnI), Bme1390I (ScrFI)/FastDigest ScrFI BmeT110I* (CYCGGG) (Bme1390I), BssKI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC), Hin6I (HinP1I)/FastDiSsiI (AciI)/FastDigest AciI (SsiI) gest HinP1I (Hin6I) (GCGC), NarI (GGCGCC) HpaII/FastDigest HpaII (CCGG), MspI (HpaII)/FastDigest MspI (CCGG) HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI (continued on next page)
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First restriction enzyme SsiI (AciI)/FastDigest AciI (SsiI)* (GCGG) SspDI (KasI) (GGCGCC) Second Restriction enzyme that cleave the newly generated recognition restriction enzyme sequence Hin1I (BsaHI)/FastDigest BsaHI (Hin1I)* (GRCGCC), Hin6I (HinP1I)/FastDiHhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I) gest HinP1I (Hin6I) (GCGC), NarI (GGCGCC) BbeI, FastDigest HaeII (BfoI), BshNI (BanI)/FastDigest BanI (BshNI), BspLI (NlaIV)/FastDigest NlaIV (BspLI), EheI (SfoI)/FastDigest EheI, HhaI/ BshNI (BanI)/FastDigest BanI (BshNI)* (GGCGCC) FastDigest HhaI, Hin1I (BsaHI)/FastDigest BsaHI (Hin1I), Hin6I (HinP1I)/ FastDigest HinP1I (Hin6I), NarI, SspDI (KasI) MaeII, SetI, TaiI (MaeII)/FastDigest TaiI TaqI/FastDigest TaqI
TaiI (MaeII)/FastDigest TaiI AatII/FastDigest AatII (GACGTC), SetI* (ACGT) (ACGT) BmeT110I* (CYCGAG), Bsp119I (BstBI)/FastDigest Bsp119I (TTCGAA), TaqI/FastDigest TaqI Bsu15I (ClaI)/FastDigest ClaI (Bsu15I) (ATCGAT), XmiI (AccI)/FastDigest (TCGA) AccI (XmiI)* (GTCGAC) EcoRI/FastDigest EcoRI (GAATTC), MunI (MfeI)/FastDigest MfeI (MunI) TasI (Tsp509I)/FastDigest (CAATTG), XapI (ApoI)/FastDigest XapI* (RAATTC), XapI (ApoI)/FastDiTsp509I (TasI) (AATT) gest XapI* (RAATTT) Acc65I (Asp718I)/FastDigest Acc65I (GGTACC), BshNI (BanI)/FastDigest BanI (BshNI)* (GGTACC), Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) TatI/FastDigest TatI* (CGTACG) (AGTACW) Bsp1407I (BsrGI)/FastDigest Bsp1407I (TGTACA) Acc65I (Asp718I)/FastDigest Acc65I (GGTACC), BshNI (BanI)/FastDigest BanI (BshNI)* (GGTACC), Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) (CGTACG) Bsp1407I (BsrGI)/FastDigest Bsp1407I (TGTACA) Tru1I (MseI)/FastDigest Tru1I (TTAA) NdeI/FastDigest NdeI (CATATG) FastDigest MseI (SaqAI) (TTAA), VspI (AseI)/FastDigest AseI (VspI) (ATTAAT) VspI (AseI)/FastDigest AseI NdeI/FastDigest NdeI (CATATG) (VspI) (ATTAAT) FastDigest MseI (SaqAI) (TTAA), Tru1I (MseI)/FastDigest Tru1I (TTAA) EcoRI /FastDigest EcoRI (GAATTC) XapI (ApoI)/FastDigest MunI (MfeI)/FastDigest MfeI (MunI) (CAATTG), TasI (Tsp509I)/FastDigest XapI* (AAATTY) Tsp509I (TasI) (AATT) EcoRI/FastDigest EcoRI (GAATTC)
TasI (Tsp509I)/FastDigest Tsp509I (TasI)
Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI, TatI/FastDigest TatI Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI Bsp1407I (BsrGI)/FastDigest Bsp1407I, Csp6I (CviQI)/FastDigest Csp6I, RsaI/FastDigest RsaI, TatI/FastDigest TatI FaiI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I FaiI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I TasI (Tsp509I)/FastDigest Tsp509I (TasI), XapI (ApoI)/FastDigest XapI TasI (Tsp509I)/FastDigest Tsp509I (TasI) EcoRI/FastDigest EcoRI, TasI (Tsp509I)/FastDigest Tsp509I (TasI), XapI (ApoI)/FastDigest XapI
TatI/FastDigest TatI* (TGTACW)
MunI (MfeI)/FastDigest MfeI (MunI) (CAATTG), TasI (Tsp509I)/FastDigest TasI (Tsp509I)/FastDigest Tsp509I (TasI) Tsp509I (TasI) (AATT) BcuI (SpeI)/FastDigest SpeI (BcuI) (ACTAGT), Eco130I (StyI)/FastDigest XbaI/FastDigest XbaI StyI (Eco130I)* (CCTAGG), NheI/FastDigest NheI (GCTAGC), XmaJI (AvrII)/ FspBI (BfaI)/FastDigest BfaI (FspBI) (TCTAGA) FastDigest AvrII (XmaJI) (CCTAGG) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) (CATG) XceI (NspI)/FastDigest NspI CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), XceI (NspI)/FastDi (XceI)* (ACATGY) PaeI (SphI)/FastDigest SphI (PaeI) (GCATGC) gest NspI (XceI) NlaIII (Hin1II) (CATG) CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II) Hin1II (NlaIII)/ FastDigest XceI (NspI)/FastDigest NspI Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), PaeI (SphI)/ (XceI)* (GCATGY) PaeI (SphI)/FastDigest SphI (PaeI) (GCATGC) FastDigest SphI (PaeI), XceI (NspI)/FastDigest NspI (XceI) AbsI (CCTCGAGG), Eco88I (AvaI)/FastDigest AvaI (Eco88I)* (CTCGAG), BmeT110I, Eco88I (AvaI)/FastDigest AvaI (Eco88I), SmoI (SmlI), TaqI/ PspXI* (VCTCGAGC), PspXI* (VCTCGAGG), PspXI* (VCTCGAGT), SmoI FastDigest TaqI, XhoI/FastDigest XhoI XhoI/FastDigest XhoI (SmlI)* (CTCGAG) (CTCGAG) TaqI/FastDigest TaqI SalI/FastDigest SalI (GTCGAC) SgrDI (CGTCGACG) Hpy99I, TaqI/FastDigest TaqI BcuI (SpeI)/FastDigest SpeI (BcuI) (ACTAGT), NheI/FastDigest NheI FspBI (BfaI)/FastDigest BfaI (FspBI) (GCTAGC), XbaI/FastDigest XbaI (TCTAGA) XmaJI (AvrII)/FastDigest BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI AvrII (XmaJI) (CCTAGG) (Eco130I), FspBI (BfaI)/FastDigest BfaI (FspBI), XmaJI (AvrII)/FastDigest Eco130I (StyI)/FastDigest StyI (Eco130I)* (CCTAGG) AvrII (XmaJI) XmiI (AccI)/FastDigest AccI CviAII (CATG) FaiI (XmiI)* (GTATAC) BmeT110I* (CY CGAG), Bsp119I (BstBI)/ FastDigest Bsp119I (TT CGAA), XmiI (AccI)/FastDigest AccI Bsu15I (ClaI)/FastDigest ClaI (Bsu15I) (ATCGAT), TaqI/FastDigest TaqI TaqI/FastDigest TaqI (XmiI)* (GTCGAC) (TCGA)
XapI (ApoI)/FastDigest XapI* (GAATTY)
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Recognition Sites Resulting from Fill-in of 5-overhang and Self-ligation

Note Single letter code
* Restriction enzymes that have degenerate recognition sequences (i.e., recognize more than one sequence). Be aware that these restriction endonucleases will cleave sequences in addition to the one listed. [ ] Enzymes that cleave the target both before and after the ligation. Fermentas FastDigest enzymes are shown in ruby. Fermentas conventional enzymes are shown in orange.
Table 1.23. Newly generated recognition sites resulting from fill-in of 5-overhang and self-ligation.
Restriction enzyme AbsI Recognition sequence CCTCGAGG Newly generated sequence Restriction enzymes that cleave after reaction the newly generated recognition sequence Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, [MnlI/FastDigest MnlI], PvuI/ CCTCGATCGAGG FastDigest PvuI, [TaqI/FastDigest TaqI] [Csp6I (CviQI)/FastDigest Csp6I], Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, Ppu21I GGTACGTACC (BsaAI)/FastDigest BsaAI (Ppu21I), [RsaI/FastDigest RsaI], SetI, TaiI (MaeII)/FastDigest TaiI ACACGCACGT [MaeII], [SetI], [TaiI (MaeII)/FastDigest TaiI] [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDiACATGCATGT gest NsiI (Mph1103I), [XceI (NspI)/FastDigest NspI (XceI)] [Bsh1236I (BstUI)/FastDigest Bsh1236I], Cac8I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDiACGCGCGCGT gest HinP1I (Hin6I), MauBI/FastDigest MauBI, PauI (BssHII)/FastDigest BssHII (PteI) ACGTGCGTGT [MaeII], [SetI], [TaiI (MaeII)/FastDigest TaiI] Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), [HpyCH4V], PaeI (SphI)/FastDiGTGCATGCAC gest SphI (PaeI), XceI (NspI)/FastDigest NspI (XceI) [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)], [BspPI (AlwI)], Bsu15I (ClaI)/FastDigest ClaI GGATCGATCC (Bsu15I), [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI CCTCATCAGC [MnlI/FastDigest MnlI] [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest ClaI (Bsu15I), TGATCGATCA [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI CCSSGG BseDI (BsaJI)/FastDigest BsaJI (BseDI) [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BseDI CCCCGG (BsaJI)/FastDigest BsaJI (BseDI), [BssKI], [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BseDI (BsaJI)/ CCGGGG FastDigest BsaJI (BseDI), [BssKI], [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] ACTAGCTAGT AluI/FastDigest AluI, CviJI, [FspBI (BfaI)/FastDigest BfaI (FspBI)], SetI CTRYATRYAG FaiI CTACATACAG FaiI CTATATATAG [FaiI] Cac8I, CviAII, FaiI, FatI, Hin1II (NlaIII)/FastDigest NlaIII (Hin1II), [HpyCH4V], PaeI (SphI)/FastDiCTGCATGCAG gest SphI (PaeI), XceI (NspI)/FastDigest NspI (XceI) CTGTATGTAG FaiI [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest ClaI (Bsu15I), AGATCGATCT [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI CCNNGG CCAAGG CCCCGG CCGGGG CCTTGG CYCGCGRG GGNNCC GGAACC GGCCCC GGGGCC GGTTCC CCTCATCAGC WCCGGCCGGW BseDI (BsaJI)/FastDigest BsaJI (BseDI) BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI (Eco130I) [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BseDI (BsaJI)/ FastDigest BsaJI (BseDI), [BssKI], [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BseDI (BsaJI)/ FastDigest BsaJI (BseDI), [BssKI], [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI (Eco130I) Bsh1236I (BstUI)/FastDigest Bsh1236I BspLI (NlaIV)/FastDigest NlaIV (BspLI) BspLI (NlaIV)/FastDigest NlaIV (BspLI) [BmgT120I], BspLI (NlaIV)/FastDigest NlaIV (BspLI), [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)], [CviJI] [BmgT120I], BspLI (NlaIV)/FastDigest NlaIV (BspLI), [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)], [CviJI] BspLI (NlaIV)/FastDigest NlaIV (BspLI) [MnlI/FastDigest MnlI] Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/ FastDigest MspI] (continued on next page)
Acc65I (Asp718I)/FastDigest Acc65I GGTACC AflIII* AflIII* AflIII* AflIII* Alw44I (ApaLI)/FastDigest ApaLI (Alw44I) BamHI/FastDigest BamHI BbvCI BclI/FastDigest BclI
ACACGT ACATGT ACGCGT ACGTGT GTGCAC GGATCC CCTCAGC TGATCA CCSGG CCCGG CCGGG ACTAGT CTRYAG CTACAG CTATAG CTGCAG CTGTAG AGATCT
BcnI (NciI)/FastDigest NciI (BcnI) BcnI (NciI)/FastDigest NciI (BcnI)* BcnI (NciI)/FastDigest NciI (BcnI)* BcuI (SpeI)/FastDigest SpeI (BcuI) BfmI (SfcI)/FastDigest SfcI (BfmI) BfmI (SfcI)/FastDigest SfcI (BfmI)* BfmI (SfcI)/FastDigest SfcI (BfmI)*
BfmI (SfcI)/FastDigest SfcI (BfmI)* BfmI (SfcI)/FastDigest SfcI (BfmI)*
BglII/FastDigest BglII Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I) Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)* Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)* Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)* Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)* BmeT110I BmgT120I BmgT120I* BmgT120I* BmgT120I* BmgT120I* Bpu10I/FastDigest Bpu10I* BsaWI
CCNGG CCAGG CCCGG CCGGG CCTGG CYCGRG GGNCC GGACC GGCCC GGGCC GGTCC CCTCAGC WCCGGW
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Table 1.23.Newly generated recognition sites resulting from fill-in of 5-overhang and self-ligation.
Restriction enzyme BseYI BshNI (BanI)/FastDigest BanI (BshNI)* Recognition sequence CCCAGC GGCACC Newly generated sequence after reaction CCCAGCCAGC GGCACGCACC GGCGCGCGCC GGTACGTACC GGTGCGTGCC ACCGGCCGGT TTCGCGAA GGGCCGGCCC TGTACGTACA GATCGATC CTTAATTAAG CCNGGCCNGG CCAGGCCAGG CCCGGCCCGG Restriction enzymes that cleave the newly generated recognition sequence [BseYI], Cac8I, CviJI Cac8I Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastDigest HinP1I (Hin6I)], PauI (BssHII)/FastDigest BssHII (PteI) [Csp6I (CviQI)/FastDigest Csp6I], Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), [RsaI/FastDigest RsaI], SetI, TaiI (MaeII)/FastDigest TaiI Cac8I Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] Bsh1236I (BstUI)/FastDigest Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest NruI (RruI) [BmgT120I], [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), [Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)], [CviJI], FseI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI) [Csp6I (CviQI)/FastDigest Csp6I], Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), [RsaI/FastDigest RsaI], SetI, TaiI (MaeII)/FastDigest TaiI [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest ClaI (Bsu15I), [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI PacI/FastDigest PacI (TTAATTAA), [FastDigest MseI (SaqAI)], TasI (Tsp509I)/FastDigest Tsp509I (TasI), [Tru1I (MseI)/FastDigest Tru1I] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] Bsh1236I (BstUI)/FastDigest Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest NruI (RruI) [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) [Bsh1236I (BstUI)/FastDigest Bsh1236I], Cac8I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I), MauBI/FastDigest MauBI, [SsiI (AciI)/FastDigest AciI (SsiI)], PauI (BssHII)/FastDigest BssHII (PteI) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDigest AciI (SsiI), TauI/FastDigest TauI [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/ FastDigest TauI [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], FseI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/ FastDigest NaeI (PdiI) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I) [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI], [SetI] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI], [SetI] Bst1107I (BstZ17I)/FastDigest BstZ17I (Bst1107I), FaiI, Hpy8I (MjaIV)/FastDigest Hpy8I, XmiI (AccI)/FastDigest AccI (XmiI) [FaiI], NdeI/FastDigest NdeI [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) AluI/FastDigest AluI, CviJI, [FspBI (BfaI)/FastDigest BfaI (FspBI)], SetI [Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)], [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], [Eco52I (EagI)/FastDigest EagI (Eco52I)], FseI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI) (continued on next page)
BshNI (BanI)/FastDigest BanI (BshNI)* GGCGCC BshNI (BanI)/FastDigest BanI (BshNI)* GGTACC BshNI (BanI)/FastDigest BanI (BshNI)* GGTGCC BshTI (AgeI)/FastDigest AgeI (BshTI) ACCGGT Bsp119I (BstBI)/FastDigest Bsp119I TTCGAA Bsp120I (PspOMI)/FastDigest Bsp120I
GGGCCC
Bsp1407I (BsrGI)/FastDigest TGTACA Bsp1407I Bsp143I (Sau3AI)/FastDigest Sau3AI GATC (Bsp143I) BspTI (AflII)/FastDigest AflII (BspTI) BssKI BssKI* BssKI* CTTAAG CCNGG CCAGG CCCGG
BssKI* BssKI*
CCGGG CCTGG
CCGGGCCGGG CCTGGCCTGG ATCGCGAT CCATGCATGG CCGCGCGCGG
Bsu15I (ClaI)/FastDigest ClaI (Bsu15I) ATCGAT BtgI* BtgI* Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I) Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)* Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I)* Cfr9I (XmaI) CCATGG CCGCGG
RCCGGY GGCCC GGGCC
RCCGGCCGGY GGCCGCCC GGGCGGCC
CCCGGG
CCCGGCCGGG
CfrI (EaeI) CpoI (RsrII)/FastDigest RsrII (CpoI)* CpoI (RsrII)/FastDigest RsrII (CpoI)* FastDigest SexAI (CsiI) FastDigest SexAI (CsiI)* FastDigest SexAI (CsiI)* Csp6I (CviQI)/FastDigest Csp6I CviAII Eco130I (StyI)/FastDigest StyI (Eco130I)* Eco130I (StyI)/FastDigest StyI (Eco130I)* Eco52I (EagI)/FastDigest EagI (Eco52I)
YGGCCR CGGACCG CGGTCCG ACCWGGT ACCAGGT ACCTGGT GTAC CATG CCATGG CCTAGG
YGGCCGGCCR CGGACGACCG CGGTCGTCCG ACCWGGCCWGGT ACCAGGCCAGGT ACCTGGCCTGGT GTATAC CATATG CCATGCATGG CCTAGCTAGG
CGGCCG
CGGCCGGCCG
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Restriction enzyme Eco81I (Bsu36I)/FastDigest Bsu36I (Eco81I)* Eco88I (AvaI)/FastDigest AvaI (Eco88I) Eco88I (AvaI)/FastDigest AvaI (Eco88I)* Eco88I (AvaI)/FastDigest AvaI (Eco88I)* Eco88I (AvaI)/FastDigest AvaI (Eco88I)* Eco88I (AvaI)/FastDigest AvaI (Eco88I)* Eco91I (BstEII)/FastDigest Eco91I Eco91I (BstEII)/FastDigest Eco91I* Eco91I (BstEII)/FastDigest Eco91I* Eco91I (BstEII)/FastDigest Eco91I* Eco91I (BstEII)/FastDigest Eco91I* EcoO109I (DraII)/FastDigest EcoO109I* EcoO109I (DraII)/FastDigest EcoO109I* EcoRI/FastDigest EcoRI EcoRII EcoRII* EcoRII* FatI
Recognition sequence CCTCAGG CYCGRG CCCGAG
Newly generated sequence Restriction enzymes that cleave after reaction the newly generated recognition sequence CCTCATCAGG CYCGRYCGRG CCCGACCGAG [MnlI/FastDigest MnlI] Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I) [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/ FastDigest TaqI] Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I) MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest TaiI MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest TaiI AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI [HphI], MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest TaiI [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), SsiI (AciI)/FastDigest AciI (SsiI), TauI/FastDigest TauI [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [CviJI], SatI (Fnu4HI)/FastDigest Fnu4HI (SatI), TauI/ FastDigest TauI PdmI (XmnI)/FastDigest PdmI, FastDigest MseI (SaqAI), [TasI (Tsp509I)/FastDigest Tsp509I (TasI)], Tru1I (MseI)/FastDigest Tru1I, VspI (AseI)/FastDigest AseI (VspI) [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDigest NsiI (Mph1103I) BfmI (SfcI)/FastDigest SfcI (BfmI), FaiI Bsh1236I (BstUI)/FastDigest Bsh1236I Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastDigest HinP1I (Hin6I)], PauI (BssHII)/FastDigest BssHII (PteI) [AluI/FastDigest AluI], BspOI (BmtI)/FastDigest BmtI (BspOI), Cac8I, [CviJI], FspBI (BfaI)/ FastDigest BfaI (FspBI), NheI/FastDigest NheI, [SetI] BseDI (BsaJI)/FastDigest BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest AciI (SsiI) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/ FastDigest MspI] AflIII, Bsh1236I (BstUI)/FastDigest Bsh1236I, MluI/FastDigest MluI MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest TaiI MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest TaiI AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI MaeII, [MaeIII], SetI, TaiI (MaeII)/FastDigest TaiI [Bsh1236I (BstUI)/FastDigest Bsh1236I], [Cac8I], [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastDigest HinP1I (Hin6I)], [MauBI/FastDigest MauBI], [PauI (BssHII)/FastDigest BssHII (PteI)] [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest ClaI (Bsu15I), [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI [Bsh1236I (BstUI)/FastDigest Bsh1236I], Cac8I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I), MauBI/FastDigest MauBI, PauI (BssHII)/FastDigest BssHII (PteI) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), [Cac8I], [Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI], [NgoMIV], [PdiI (NaeI)/ FastDigest NaeI (PdiI)] AanI (PsiI)/FastDigest PsiI (AanI), FaiI BseDI (BsaJI)/FastDigest BsaJI (BseDI), Bsh1236I (BstUI)/FastDigest Bsh1236I, BtgI, Cfr42I (SacII), MspA1I, SsiI (AciI)/FastDigest AciI (SsiI) FastDigest MseI (SaqAI), [TasI (Tsp509I)/FastDigest Tsp509I (TasI)], Tru1I (MseI)/FastDigest Tru1I, VspI (AseI)/FastDigest AseI (VspI) BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI (Eco130I) BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI (Eco130I) BseDI (BsaJI)/FastDigest BsaJI (BseDI), Eco130I (StyI)/FastDigest StyI (Eco130I) (continued on next page)
CCCGGG
CCCGGCCGGG
CTCGAG CTCGGG GGTNACC GGTAACC GGTCACC GGTGACC GGTTACC RGGCCCY RGGGCCY GAATTC CCWGG CCAGG CCTGG CATG
CTCGATCGAG CTCGGTCGGG GGTNACGTNACC GGTAACGTAACC GGTCACGTCACC GGTGACGTGACC GGTTACGTTACC RGGCCGCCCY RGGGCGGCCY GAATTAATTC CCWGGCCWGG CCAGGCCAGG CCTGGCCTGG CATGCATG CTATAG GRCGCGYC GCGCGC AAGCTAGCTT CCGCGG TCCGGCCGGA ACGCGT GTNACGTNAC GTAACGTAAC GTCACGTCAC GTGACGTGAC GTTACGTTAC CGCGCGCGCGCG GATCGATC ACGCGCGCGT
FspBI (BfaI)/FastDigest BfaI (FspBI) CTAG Hin1I (BsaHI)/FastDigest BsaHI (Hin1I) GRCGYC Hin6I (HinP1I)/FastDigest HinP1I GCGC (Hin6I) HindIII/FastDigest HindIII
AAGCTT CCGG TCCGGA ACGT GTNAC GTAAC GTCAC GTGAC GTTAC CGCGCGCG GATC ACGCGT
HpaII/FastDigest HpaII Kpn2I (BspEI)/FastDigest Kpn2I MaeII MaeIII MaeIII* MaeIII* MaeIII* MaeIII* MauBI/FastDigest MauBI MboI/FastDigest MboI MluI/FastDigest MluI
MreI (Sse232I)/FastDigest MreI FastDigest MseI (SaqAI) MspI (HpaII)/FastDigest MspI MunI (MfeI)/FastDigest MfeI (MunI) MvaI (BstNI)/FastDigest MvaI MvaI (BstNI)/FastDigest MvaI* MvaI (BstNI)/FastDigest MvaI*
CGCCGGCG TTAA CCGG CAATTG CCWGG CCAGG CCTGG
CGCCGGCCGGCG TTATAA CCGCGG CAATTAATTG CCWWGG CCAAGG CCTTGG
www.fermentas.com
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Restriction enzyme NarI NcoI/FastDigest NcoI NdeI/FastDigest NdeI NgoMIV Recognition sequence GGCGCC CCATGG CATATG GCCGGC Newly generated sequence Restriction enzymes that cleave after reaction the newly generated recognition sequence Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastDiGGCGCGCC gest HinP1I (Hin6I)], SgsI (AscI)/FastDigest AscI (SgsI), PauI (BssHII)/FastDigest BssHII (PteI) [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDiCCATGCATGG gest NsiI (Mph1103I) CATATATG [FaiI] Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), [Cac8I], [Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest GCCGGCCGGC EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI], [NgoMIV], [PdiI (NaeI)/ FastDigest NaeI (PdiI)] AluI/FastDigest AluI, [BspOI (BmtI)/FastDigest BmtI (BspOI)], [Cac8I], CviJI, [FspBI (BfaI)/ GCTAGCTAGC FastDigest BfaI (FspBI)], [NheI/FastDigest NheI], SetI GTSACGTSAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI GTCACGTCAC AjiI (BmgBI), MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI GTGACGTGAC MaeII, [MaeIII], [NmuCI (Tsp45I)/FastDigest NmuCI], SetI, TaiI (MaeII)/FastDigest TaiI [Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I)], [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], Cac8I, Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I), [CfrI (EaeI)], [CviJI], [Eco52I (EagI)/FastDigest GCGGCCGGCCGC EagI (Eco52I)], FseI, HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI, NgoMIV, PdiI (NaeI)/FastDigest NaeI (PdiI), [SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)], [SsiI (AciI)/FastDigest AciI (SsiI)], [TauI/FastDigest TauI] [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDiTCATGCATGA gest NsiI (Mph1103I) CCCAGCAGGG BseYI, EcoP15I [Bsh1236I (BstUI)/FastDigest Bsh1236I], [Cac8I], [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastGCGCGCGCGC Digest HinP1I (Hin6I)], MauBI/FastDigest MauBI, [PauI (BssHII)/FastDigest BssHII (PteI)] [Csp6I (CviQI)/FastDigest Csp6I], Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, [Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II)], Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), [RsaI/FastDiCGTACGTACG gest RsaI], SetI, TaiI (MaeII)/FastDigest TaiI [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII TCCNGGCCNGGA (BsuRI), CviJI [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII TCCAGGCCAGGA (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest TCCCGGCCCGGA Sau96I (Cfr13I), CviJI, [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [BcnI (NciI)/FastDigest NciI (BcnI)], [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], TCCGGGCCGGGA BmgT120I, [BssKI], BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), Cfr13I (Sau96I)/FastDigest Sau96I (Cfr13I), CviJI, [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] [Bme1390I (ScrFI)/FastDigest ScrFI (Bme1390I)], [BssKI], BsuRI (HaeIII)/FastDigest HaeIII TCCTGGCCTGGA (BsuRI), CviJI, [EcoRII], [MvaI (BstNI)/FastDigest MvaI] [CviAII], [FaiI], [FatI], [Hin1II (NlaIII)/FastDigest NlaIII (Hin1II)], HpyCH4V, Mph1103I (NsiI)/FastDiACATGCATGT gest NsiI (Mph1103I), [XceI (NspI)/FastDigest NspI (XceI)] AACGCGTT VCTCGATCGAGB RGATCGATCY GACNNNNGTC GACNAANGTC GACNCCNGTC GACNGGNGTC GACNTTNGTC GTCGATCGAC GGGACGACCC GCNNGC GCAAGC GCCCGC GCGGGC GCTTGC CRCCGGCCGGYG AflIII, Bsh1236I (BstUI)/FastDigest Bsh1236I, MluI/FastDigest MluI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/ FastDigest TaqI] [Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I)], Bsu15I (ClaI)/FastDigest ClaI (Bsu15I), [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI BoxI (PshAI)/FastDigest PshAI (BoxI) BoxI (PshAI)/FastDigest PshAI (BoxI) BoxI (PshAI)/FastDigest PshAI (BoxI) BoxI (PshAI)/FastDigest PshAI (BoxI) BoxI (PshAI)/FastDigest PshAI (BoxI) Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/ FastDigest TaqI] [FaqI (BsmFI)/FastDigest BsmFI (FaqI)] Cac8I Cac8I Cac8I, SmuI (FauI), [SsiI (AciI)/FastDigest AciI (SsiI)] Cac8I Cac8I Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), BsuRI (HaeIII)/FastDigest HaeIII (BsuRI), [Cfr10I (BsrFI)/FastDigest BsrFI (Cfr10I)], CfrI (EaeI), CviJI, Eco52I (EagI)/FastDigest EagI (Eco52I), [HpaII/FastDigest HpaII], [MspI (HpaII)/FastDigest MspI] (continued on next page)
NheI/FastDigest NheI NmuCI (Tsp45I)/FastDigest NmuCI NmuCI (Tsp45I)/FastDigest NmuCI* NmuCI (Tsp45I)/FastDigest NmuCI*
GCTAGC GTSAC GTCAC GTGAC
NotI/FastDigest NotI
GCGGCCGC
PagI (BspHI)/FastDigest BspHI (PagI) TCATGA PasI*
CCCAGGG
PauI (BssHII)/FastDigest BssHII (PteI) GCGCGC Pfl23II (BsiWI)/FastDigest BsiWI (Pfl23II) PfoI/FastDigest PfoI PfoI/FastDigest PfoI* PfoI/FastDigest PfoI* CGTACG TCCNGGA TCCAGGA TCCCGGA
PfoI/FastDigest PfoI* PfoI/FastDigest PfoI* PscI (PciI) Psp1406I (AclI)/FastDigest AclI (Psp1406I)
TCCGGGA TCCTGGA ACATGT AACGTT VCTCGAGB RGATCY GACNNNGTC GACNANGTC GACNCNGTC GACNGNGTC GACNTNGTC GTCGAC GGGACCC GCNGC GCAGC GCCGC GCGGC GCTGC CRCCGGYG
PspXI PsuI (BstYI)/FastDigest PsuI PsyI (Tth111I)/FastDigest PsyI PsyI (Tth111I)/FastDigest PsyI* PsyI (Tth111I)/FastDigest PsyI* PsyI (Tth111I)/FastDigest PsyI* PsyI (Tth111I)/FastDigest PsyI* SalI/FastDigest SalI FastDigest SanDI (KflI)* SatI (Fnu4HI)/FastDigest Fnu4HI (SatI) SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)* SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)* SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)* SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)* SgrAI
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Restriction enzyme SgrDI SgsI (AscI)/FastDigest AscI (SgsI) SmoI (SmlI)* SmoI (SmlI)* SsiI (AciI)/FastDigest AciI (SsiI) SspDI (KasI) TaqI/FastDigest TaqI TasI (Tsp509I)/FastDigest Tsp509I (TasI)
Recognition sequence CGTCGACG GGCGCGCC CTCGAG CTTAAG CCGC GGCGCC TCGA AATT WGTACW TTAA GCWGC GCAGC GCTGC ATTAAT RAATTY TCTAGA CTCGAG CCTAGG GTATAC GTCGAC GTCTAC
TatI/FastDigest TatI Tru1I (MseI)/FastDigest Tru1I TseI TseI* TseI* VspI (AseI)/FastDigest AseI (VspI) XapI (ApoI)/FastDigest XapI XbaI/FastDigest XbaI
XhoI/FastDigest XhoI
XmaJI (AvrII)/FastDigest AvrII (XmaJI) XmiI (AccI)/FastDigest AccI (XmiI)* XmiI (AccI)/FastDigest AccI (XmiI)* XmiI (AccI)/FastDigest AccI (XmiI)*
Newly generated sequence Restriction enzymes that cleave after reaction the newly generated recognition sequence Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, [Hpy99I], MboI/FastDigest MboI, PvuI/FastDigest PvuI, CGTCGATCGACG [TaqI/FastDigest TaqI] [Bsh1236I (BstUI)/FastDigest Bsh1236I], [Cac8I], [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastGGCGCGCGCGCC Digest HinP1I (Hin6I)], MauBI/FastDigest MauBI, [PauI (BssHII)/FastDigest BssHII (PteI)] Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/ CTCGATCGAG FastDigest TaqI] PacI/FastDigest PacI (TTAATTAA), [FastDigest MseI (SaqAI)], TasI (Tsp509I)/FastDigest CTTAATTAAG Tsp509I (TasI), [Tru1I (MseI)/FastDigest Tru1I] Bsh1236I (BstUI)/FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest CCGCGC HinP1I (Hin6I), [SsiI (AciI)/FastDigest AciI (SsiI)] Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, [HhaI/FastDigest HhaI], [Hin6I (HinP1I)/FastGGCGCGCGCC Digest HinP1I (Hin6I)], PauI (BssHII)/FastDigest BssHII (PteI) TCGCGA Bsh1236I (BstUI)/FastDigest Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest NruI (RruI) FastDigest MseI (SaqAI), [TasI (Tsp509I)/FastDigest Tsp509I (TasI)], Tru1I (MseI)/FastDigest AATTAATT Tru1I, VspI (AseI)/FastDigest AseI (VspI) [Csp6I (CviQI)/FastDigest Csp6I], Eco105I (SnaBI)/FastDigest SnaBI (Eco105I), MaeII, Ppu21I WGTACGTACW (BsaAI)/FastDigest BsaAI (Ppu21I), [RsaI/FastDigest RsaI], SetI, TaiI (MaeII)/FastDigest TaiI TTATAA AanI (PsiI)/FastDigest PsiI (AanI), FaiI GCWGCWGC [SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)], [TseI] [BseXI (BbvI)/FastDigest BseXI], EcoP15I, [Lsp1109I (BbvI)/FastDigest BbvI (Lsp1109I)], [SatI GCAGCAGC (Fnu4HI)/FastDigest Fnu4HI (SatI)], [TseI] GCTGCTGC [SatI (Fnu4HI)/FastDigest Fnu4HI (SatI)], [TseI] ATTATAAT AanI (PsiI)/FastDigest PsiI (AanI), FaiI FastDigest MseI (SaqAI), [TasI (Tsp509I)/FastDigest Tsp509I (TasI)], Tru1I (MseI)/FastDigest RAATTAATTY Tru1I, VspI (AseI)/FastDigest AseI (VspI) TCTAGCTAGA AluI/FastDigest AluI, CviJI, [FspBI (BfaI)/FastDigest BfaI (FspBI)], SetI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I), Bsp143I (Sau3AI)/FastDigest Sau3AI (Bsp143I), DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/ CTCGATCGAG FastDigest TaqI] CCTAGCTAGG AluI/FastDigest AluI, CviJI, [FspBI (BfaI)/FastDigest BfaI (FspBI)], SetI GTATATAC [FaiI] GTCGCGAC Bsh1236I (BstUI)/FastDigest Bsh1236I, Hpy188III, Bsp68I (NruI)/FastDigest NruI (RruI) GTCTCTAC Alw26I (BsmAI)/FastDigest Alw26I
Recognition Sites Resulting from Removal of 3-overhang and Self-ligation

Table 1.24. Newly generated recognition sites resulting from removal of 3-overhang and self-ligation.
Restriction enzyme AasI (DrdI)/FastDigest DrdI (AasI) AdeI (DraIII)/FastDigest DraIII (AdeI) AgsI BglI/FastDigest BglI Bsh1285I (BsiEI)/FastDigest BsiEI (Bsh1285I) CaiI (AlwNI)/FastDigest AlwNI (CaiI) Cfr42I (SacII) Eam1105I (AhdI)/FastDigest Eam1105I FseI Hpy188I PacI/FastDigest PacI (TTAATTAA) PvuI/FastDigest PvuI SdaI (SbfI)/FastDigest SbfI (SdaI) SfaAI (AsiSI)/FastDigest AsiSI (SfaAI) SfiI/FastDigest SfiI TaaI (HpyCH4III)/FastDigest TaaI Recognition sequence GACNNNNNNGTC CACNNNGTG TTSAA GCCNNNNNGGC CGRYCG CAGNNNCTG CCGCGG GACNNNNNGTC GGCCGGCC TCNGA TTAATTAA CGATCG CCTGCAGG GCGATCGC GGCCNNNNNGGCC ACNGT Newly generated sequence after reaction GACNNNNGTC CACGTG TTAA GCCNNGGC CGCG CAGCTG CCGG GACNNNNGTC GGCC TCGA TTATAA CGCG CCGG GCGCGC GGCCNNGGCC ACGT Restriction enzymes that cleave the newly generated recognition sequence BoxI (PshAI)/FastDigest PshAI (BoxI) Eco72I (PmlI)/FastDigest PmlI (Eco72I), MaeII, Ppu21I (BsaAI)/FastDigest BsaAI (Ppu21I), SetI, TaiI (MaeII)/FastDigest TaiI FastDigest MseI (SaqAI), Tru1I (MseI)/FastDigest Tru1I BseDI (BsaJI)/FastDigest BsaJI (BseDI) Bsh1236I (BstUI)/FastDigest Bsh1236I AluI/FastDigest AluI, CviJI, MspA1I, PvuII/FastDigest PvuII, SetI HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI BoxI (PshAI)/FastDigest PshAI (BoxI) [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [CviJI] TaqI/FastDigest TaqI AanI (PsiI)/FastDigest PsiI (AanI), FaiI Bsh1236I (BstUI)/FastDigest Bsh1236I HpaII/FastDigest HpaII, MspI (HpaII)/FastDigest MspI Bsh1236I (BstUI)/FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I (HinP1I)/FastDigest HinP1I (Hin6I), PauI (BssHII)/FastDigest BssHII (PteI) BseDI (BsaJI)/FastDigest BsaJI (BseDI), [BsuRI (HaeIII)/FastDigest HaeIII (BsuRI)], [CviJI] MaeII, SetI, TaiI (MaeII)/FastDigest TaiI
Note
Fermentas FastDigest enzymes are shown in ruby. Fermentas conventional enzymes are shown in orange.
Single letter code

R = G or A; Y = C or T; W = A or T; K = G or T; S = C or G; H = A, C or T; B = C, G or T; D = A, G or T; N = G, A, T or C. M = A or C V = A, C or G;
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Selection Guides
Alphabetic List of Commercially Available Restriction Enzymes
Note
[ ] Enzymes have different cleavage specificities (neoschizomers). m Isoschizomers with different sensitivity to methylation. * Restriction enzymes produced by Fermentas are shown in bold. Fermentas FastDigest enzymes are shown in ruby. Fermentas Conventional enzymes are shown in orange. Table 1.25. Commercially available restriction enzymes.
Enzyme AanI AarI AasI AatI AatII AbsI Acc16I Acc36I Acc65I AccB1I AccB7I AccBSI AccI AccII AccIII AciI AclI AclWI AcoI AcsI AcuI AcvI AcyI AdeI AfaI AfeI AfiI AflII AflIII AgeI AgsI AhdI AhlI AjiI AjnI AjuI AleI AlfI AloI AluI AluBI Alw21I Alw26I Alw44I AlwI AlwNI Ama87I Aor13HI Aor51HI ApaI Specificity 53 TTATAA CACCTGC(4/8) GACNNNNNNGTC AGGCCT GACGTC CCTCGAGG TGCGCA ACCTGC(4/8) GGTACC GGYRCC CCANNNNNTGG CCGCTC(-3/-3) GTMKAC CGCG TCCGGA CCGC(-3/-1) AACGTT GGATC(4/5) YGGCCR RAATTY CTGAAG(16/14) CACGTG GRCGYC CACNNNGTG GTAC AGCGCT CCNNNNNNNGG CTTAAG ACRYGT ACCGGT TTSAA GACNNNNNGTC ACTAGT CACGTC(-3/-3) CCWGG (6/11)CCAA(N)7TTC(12/7) CACNNNNGTG (10/12)GCA(N)6TGC(12/10) (7/12)GAAC(N)6TCC(12/7) AGCT AGCT GWGCWC GTCTC(1/5) GTGCAC GGATC(4/5) CAGNNNCTG CYCGRG TCCGGA AGCGCT GGGCCC FastDigest enzyme FastDigest PsiI (AanI) FastDigest DrdI (AasI) FastDigest StuI (Eco147I) FastDigest AatII FastDigest FspI (NsbI) FastDigest BspMI (BveI) FastDigest Acc65I, [FastDigest KpnIm](GGTACC) FastDigest BanI (BshNI) FastDigest PflMI (Van91I) FastDigest BsrBI (MbiI) FastDigest AccI (XmiI) FastDigest Bsh1236I FastDigest Kpn2Im FastDigest AciI (SsiI) FastDigest AclI (Psp1406I) Page Conventional enzyme 55 34 64 11 39 29 11 43 19 54 30 11 25 44 12 12 AanI (PsiI) AarI AasI (DrdI) Eco147I (StuI) AatII NsbI (FspI) BveI (BspMI) Acc65I (Asp718I), [KpnIm](GGTACC) BshNI (BanI) Van91I (PflMI) MbiI (BsrBI) XmiI (AccI) Bsh1236I (BstUI) Kpn2I (BspEI)m SsiI (AciI) Psp1406I (AclI) BspPI (AlwI) CfrI (EaeI) XapI (ApoI) Eco57I (AcuI) Eco72I (PmlI) Hin1I (BsaHI) AdeI (DraIII) [Csp6I (CviQI)m](GTAC), RsaI Eco47III (AfeI) BseLI (BslI) BspTI (AflII) BshTI (AgeI) Eam1105I (AhdI) BcuI (SpeI) AjiI (BmgBI) EcoRIIm, [MvaI (BstNI)](CCWGG) AjuI OliI (AleI) AlfI AloI AluI AluI Alw21I (BsiHKAI) Alw26I (BsmAI) Alw44I (ApaLI) BspPI (AlwI) CaiI (AlwNI) Eco88I (AvaI) Kpn2I (BspEI) Eco47III (AfeI) ApaI, [Bsp120I (PspOMI)](GGGCCC) Page 80 80 80 115 81 133 104 81 124 97 154 126 157 97 124 149 139 101 105 155 112 113 120 82 107 141 111 95 101 98 109 88 82 116 130 83 134 83 84 84 84 84 85 85 101 104 113 124 111 85 99 Commercially available isoschizomers from other vendors PsiI DrdI, DseDI PceI, SseBI, StuI [ZraIm] AviII, FspI BfuAI, BspMI Asp718Im BanI, BspT107I BasI, PflMI BsrBI AccI, FblI BspFNI, BstFNI, BstUI, MvnI Aor13HI, BseAIm, Bsp13I, BspEIm, MroIm AciI, BspACI AclI AlwI EaeI ApoI AcuI BbrPI, PmaCI, PmlI, PspCI BsaHI, BssNI, BstACI, Hsp92I DraIII [CviQIm], [RsaNI] AfeI, Aor51HI Bsc4I, BslI AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464I AgeI, AsiGI, CspAIm, PinAI AspEI, BmeRI, DriI, EclHKI SpeI BmgBI, BtrI [BseBI], [Bst2UI], [BstNI], [BstOI], Psp6I, PspGIm AleI
1
Single letter code
FastDigest XapI FastDigest AcuI (Eco57I) FastDigest PmlI (Eco72I) FastDigest BsaHI (Hin1I) FastDigest DraIII (AdeI) [FastDigest Csp6Im](GTAC), FastDigest RsaI FastDigest AfeI (Eco47III) FastDigest BslI (BseLI) FastDigest AflII (BspTI) FastDigest AgeI (BshTI) FastDigest Eam1105I FastDigest SpeI (BcuI) [FastDigest MvaI](CCWGG) FastDigest AjuI FastDigest AleI (OliI)
68 12 54 24 34 32 58 13 26 13 13 35 63 48 14 14
FastDigest AluI FastDigest AluI FastDigest Alw21I FastDigest Alw26I FastDigest ApaLI (Alw44I) FastDigest AlwNI (CaiI) FastDigest AvaI (Eco88I) FastDigest Kpn2I FastDigest AfeI (Eco47III) FastDigest ApaI, [FastDigest Bsp120I](GGGCCC)
14 14 15 15 16 15 17 44 13 16 28
AluBI Bbv12I, BsiHKAI BsmAIm, BstMAI ApaLIm, VneI AclWI AlwNI AvaI, [BmeT110I], BsiHKCI, BsoBI AccIII, BseAI, Bsp13I, BspEI, MroI AfeI [PspOMI] (continued on next page)
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Table 1.25.Commercially available restriction enzymes.
1
Enzyme ApaLI ApeKI ApoI ArsI AscI AseI AsiGI AsiSI Asp700I Asp718I AspA2I AspEI AspI AspLEI AspS9I AssI AsuC2I AsuHPI AsuII AsuNHI AvaI AvaII AviII AvrII AxyI BaeGI BaeI BalI BamHI BanI BanII BanIII BarI BasI BauI BbeI BbrPI BbsI BbuI Bbv12I BbvCI BbvI BccI BceAI BcgI BciVI BclI BcnI BcuI BdaI BfaI BfiI BfmI BfrI BfuAI BfuCI BfuI BglI
Specificity 53 GTGCAC GCWGC RAATTY (8/13)GAC(N)6TTYG(11/6) GGCGCGCC ATTAAT ACCGGT GCGATCGC GAANNNNTTC GGTACC CCTAGG GACNNNNNGTC GACNNNGTC GCGC GGNCC AGTACT CCSGG GGTGA(8/7) TTCGAA GCTAGC CYCGRG GGWCC TGCGCA CCTAGG CCTNAGG GKGCMC (10/15)AC(N)4GTAYC(12/7) TGGCCA GGATCC GGYRCC GRGCYC ATCGAT (7/12)GAAG(N)6TAC(12/7) CCANNNNNTGG CACGAG(-5/-1) GGCGCC CACGTG GAAGAC(2/6) GCATGC GWGCWC CCTCAGC(-5/-2) GCAGC(8/12) CCATC(4/5) ACGGC(12/14) (10/12)CGA(N)6TGC(12/10) GTATCC(6/5) TGATCA CCSGG ACTAGT (10/12)TGA(N)6TCA(12/10) CTAG ACTGGG(5/4) CTRYAG CTTAAG ACCTGC(4/8) GATC GTATCC(6/5) GCCNNNNNGGC
FastDigest enzyme FastDigest ApaLI (Alw44I)m FastDigest XapI FastDigest AscI (SgsI) FastDigest AseI (VspI) FastDigest AgeI (BshTI) FastDigest AsiSI (SfaAI) FastDigest PdmI FastDigest Acc65Im, [FastDigest KpnIm](GGTACC) FastDigest AvrII (XmaJI) FastDigest Eam1105I FastDigest PsyI FastDigest HhaI, [FastDigest HinP1I (Hin6I)](GCGC) FastDigest Sau96I (Cfr13I) FastDigest ScaI FastDigest NciI (BcnI) FastDigest Bsp119Im [FastDigest BmtI (BspOI)](GCTAGC), FastDigest NheI FastDigest AvaI (Eco88I)m FastDigest AvaII (Eco47I) FastDigest FspI (NsbI) FastDigest AvrII (XmaJI) FastDigest Bsu36I (Eco81I) FastDigest Bme1580I (BseSI)
Page Conventional enzyme 16 68 16 17 13 17 53 11 43 18 35 57 40 42 60 61 49 27 22 50 17 18 39 18 32 21 47 18 19 32 54 38 54 19 64 15 19 25 Alw44I (ApaLI)m XapI (ApoI) SgsI (AscI) VspI (AseI) BshTI (AgeI) SfaAI (AsiSI) PdmI (XmnI) Acc65I (Asp718I)m, [KpnIm](GGTACC) XmaJI (AvrII) Eam1105I (AhdI) PsyI (Tth111I) HhaI, [Hin6I (HinP1I)](GCGC) Cfr13I (Sau96I) ScaI BcnI (NciI) HphI Bsp119I (BstBI)m [BspOI (BmtI)](GCTAGC), NheI Eco88I (AvaI)m Eco47I (AvaII) NsbI (FspI) XmaJI (AvrII) Eco81I (Bsu36I) BseSI (Bme1580I) MlsI (MscI) BamHI BshNI (BanI) Eco24I (BanII) Bsu15I (ClaI) Van91I (PflMI) BauI (BssSI) [EheI (SfoI)](GGCGCC), [SspDI (KasI)](GGCGCC) Eco72I (PmlI) BpiI (BbsI) PaeI (SphI) Alw21I (BsiHKAI) Lsp1109I (BbvI), BseXI (BbvI)
Page 85 155 147 154 98 145 136 81 124 156 109 140 120 121 106 144 88 123 99 100 132 113 111 133 156 113 96 128 86 97 110 103 154 87 117 150 113 92 134 84 125 96
Commercially available isoschizomers from other vendors ApaLI, VneI TseI AcsI AscI, PalAI AseI, PshBI AgeI, CspAI, PinAI AsiSI, RgaI, SgfI MroXI, XmnI
AvrII, BlnI AhdI, BmeRI, DriI, EclHKI PflFI, Tth111I BstHHI, CfoI, [HinP1I], [HspAI] [BmgT120I], PspPI, Sau96I BmcAI, ZrmI BpuMI, NciI Bpu14I, BspT104Im, BstBIm, Csp45I, NspVm, SfuI [BmtI] Ama87I, AvaI, [BmeT110Im], BsiHKCI, BsoBIm AvaII, Bme18I, SinI, VpaK11BI Acc16I, FspI AspA2I, AvrII, BlnI Bse21I, Bsu36I BstSLI MluNI, MscI, Msp20I AccB1I, BanI, BspT107I EcoT38I, FriOI Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI AccB7I, PflMI BssSI, Bst2BI [DinI], [EgeI], [KasI], [Mly113I], [NarI], [SfoIm] AcvI, PmaCI, PmlI, PspCI BbsI, BpuAI, BstV2I SphI BsiHKAI BbvI, BstV1I
FastDigest MscI (MlsI) FastDigest BamHI FastDigest BanI (BshNI) FastDigest ClaI (Bsu15I) FastDigest PflMI (Van91I) [FastDigest EheI](GGCGCC) FastDigest PmlI (Eco72I) FastDigest BbsI (BpiI) FastDigest SphI (PaeI) FastDigest Alw21I
FastDigest BbvI (Lsp1109I), FastDigest BseXI
FastDigest BclI FastDigest NciI (BcnI) FastDigest SpeI (BcuI) FastDigest BfaI (FspBI) FastDigest SfcI (BfmI) FastDigest AflII (BspTI) FastDigest BspMI (BveI) FastDigest Sau3AI (Bsp143I), FastDigest MboIm FastDigest BglI
20 49 63 20 62 13 29 60 44 20
BfuI (BciVI) BclI BcnI (NciI) BcuI (SpeI) BdaI FspBI (BfaI) BfiI (BmrI) BfmI (SfcI) BspTI (AflII) BveI (BspMI) Bsp143I (Sau3AI), MboIm BfuI (BciVI) BglI
90 87 88 88 88 119 89 89 101 104 99 127 90 90
FbaI, Ksp22I AsuC2I, BpuMI, NciI AhlI, SpeI BfaI, MaeI, XspI BmrI, BmuI BstSFI, SfcI AflII, Bst98I, BstAFI, MspCI, Vha464I Acc36I, BspMI BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm, Sau3AIm BciVI (continued on next page)
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Enzyme BglII BisI BlnI BlpI BlsI BmcAI Bme1390I Bme1580I Bme18I BmeRI BmeT110I BmgBI BmgT120I BmiI BmrFI BmrI BmtI BmuI BoxI BpiI BplI BpmI Bpu10I Bpu1102I Bpu14I BpuAI BpuEI BpuMI BpvUI Bsa29I BsaAI BsaBI BsaHI BsaI BsaJI BsaMI BsaWI BsaXI Bsc4I Bse118I Bse1I Bse21I Bse3DI Bse8I BseAI BseBI BseCI BseDI BseGI BseJI BseLI BseMI BseMII BseNI BsePI BseRI BseSI BseXI BseX3I BseYI Specificity 53 AGATCT GCNGC CCTAGG GCTNAGC GCNGC AGTACT CCNGG GKGCMC GGWCC GACNNNNNGTC CYCGRG CACGTC(-3/-3) GGNCC GGNNCC CCNGG ACTGGG(5/4) GCTAGC ACTGGG(5/4) GACNNNNGTC GAAGAC(2/6) (8/13)GAG(N)5CTC(13/8) CTGGAG(16/14) CCTNAGC(-5/-2) GCTNAGC TTCGAA GAAGAC(2/6) CTTGAG(16/14) CCSGG CGATCG ATCGAT YACGTR GATNNNNATC GRCGYC GGTCTC(1/5) CCNNGG GAATGC(1/-1) WCCGGW (9/12)AC(N)5CTCC(10/7) CCNNNNNNNGG RCCGGY ACTGG(1/-1) CCTNAGG GCAATG(2/0) GATNNNNATC TCCGGA CCWGG ATCGAT CCNNGG GGATG(2/0) GATNNNNATC CCNNNNNNNGG GCAATG(2/0) CTCAG(10/8) ACTGG(1/-1) GCGCGC GAGGAG(10/8) GKGCMC GCAGC(8/12) CGGCCG CCCAGC(-5/-1) FastDigest enzyme FastDigest BglII FastDigest AvrII (XmaJI) FastDigest BlpI (Bpu1102I) FastDigest ScaI FastDigest ScrFI (Bme1390I)
Page Conventional enzyme 21 18 21 61 61 21 18 35 17 BglII XmaJI (AvrII) Bpu1102I (BlpI) ScaI Bme1390I (ScrFI) BseSI (Bme1580I) Eco47I (AvaII) Eam1105I (AhdI) [Eco88I (AvaI)m](CYCGRG) AjiI (BmgBI) [Cfr13I (Sau96I)](GGNCC) BspLI (NlaIV) Bme1390I (ScrFI) BfiI (BmrI) BspOI (BmtI), [NheIm](GCTAGC) BfiI (BmrI) BoxI (PshAI) BpiI (BbsI) BplI GsuI (BpmI)m Bpu10I Bpu1102I (BlpI) Bsp119I (BstBI) BpiI (BbsI) BcnI (NciI) PvuI Bsu15I (ClaI) Ppu21I (BsaAI) BseJI (BsaBI) Hin1I (BsaHI)m Eco31I (BsaI) BseDI (BsaJI) Mva1269I (BsmI)
Page 90 156 93 144 91 96 111 109 113 82 106 100 91 89 100 132 89 91 92 92 119 93 93 99 92 88 141 103 138 94 120 110 94 131
Commercially available isoschizomers from other vendors [BlsI], GluI AspA2I, AvrII BlpI, Bsp1720I, CelII [BisI], [GluI] AssI, ZrmI BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I] BaeGI, BstSLI AvaII, SinI, VpaK11BI AhdI, AspEI, DriI, EclHKI [Ama87I], [AvaIm], [BsiHKCI], [BsoBI] BtrI [AspS9I], [PspPI], [Sau96I] NlaIV, PspN4I [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I] BmuI [AsuNHI], BmtI BmrI BstPAI, PshAI BbsI, BpuAI, BstV2I BpmI BlpI, Bsp1720I, CelII AsuII, BspT104I, BstBI, Csp45I, NspV, SfuI BbsI, BstV2I AsuC2I, NciI MvrI, Ple19I BanIII, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI BsaAI, BstBAI BsaBI, Bse8I AcyI, BsaHI, BssNI, BstACI, Hsp92I Bso31I, BspTNI BsaJI, BssECI BsmI, PctI
FastDigest Bme1580I (BseSI) FastDigest AvaII (Eco47I) FastDigest Eam1105I [FastDigest AvaI (Eco88I)m](CYCGRG)
[FastDigest Sau96I (Cfr13I)](GGNCC) 60 51 FastDigest NlaIV (BspLI) 61 FastDigest ScrFI (Bme1390I) FastDigest BmtI (BspOI), [FastDigest NheIm](GCTAGC) FastDigest PshAI (BoxI) FastDigest BbsI (BpiI) FastDigest BplI FastDigest BpmI (GsuI)m FastDigest Bpu10I FastDigest BlpI (Bpu1102I) FastDigest Bsp119I FastDigest BbsI (BpiI) FastDigest NciI (BcnI) FastDigest PvuI FastDigest ClaI (Bsu15I) FastDigest BsaAI (Ppu21I) FastDigest BsaBI (BseJI) FastDigest BsaHI (Hin1I)m FastDigest Eco31I FastDigest BsaJI (BseDI) FastDigest Mva1269I 22 50 55 19 22 22 23 21 27 19 49 57 32 23 23 24 36 24 49
FastDigest BslI (BseLI) FastDigest BsrFI (Cfr10I) FastDigest BseNI FastDigest Bsu36I (Eco81I) FastDigest BsrDI (BseMI) FastDigest BsaBI (BseJI) FastDigest Kpn2Im FastDigest MvaI FastDigest ClaI (Bsu15I) FastDigest BsaJI (BseDI) FastDigest BseGI, [FastDigest FokI](GGATG(9/13)) FastDigest BsaBI (BseJI) FastDigest BslI (BseLI) FastDigest BsrDI (BseMI) FastDigest BspCNI (BseMII) FastDigest BseNI FastDigest BssHII (PteI)
26 30 25 32 30 23 44 48 32 24 24 38 23 26 30 29 25 31 21 25 19 34 56
BseLI (BslI) Cfr10I (BsrFI) BseNI (BsrI) Eco81I (Bsu36I) BseMI (BsrDI) BseJI (BsaBI) Kpn2I (BspEI)m [EcoRII](CCWGG), MvaI (BstNI) Bsu15I (ClaI) BseDI (BsaJI) BseGI (BtsCI) BseJI (BsaBI) BseLI (BslI) BseMI (BsrDI) BseMII (BspCNI) BseNI (BsrI) PauI (BssHII) BseSI (Bme1580I) BseXI (BbvI), Lsp1109I (BbvI) Eco52I (EagI)
95 105 96 113 95 94 124 116 130 103 94 94 94 95 95 95 96 135 96 96 125 111
AfiI, BslI BsrFI, BssAI BsrI, BsrSI AxyI, Bsu36I BsrDI BsaBI AccIIIm, Aor13HI, Bsp13I, BspEIm, MroIm [AjnI], Bst2UI, BstNI, BstOI, [Psp6I], [PspGI] BanIII, Bsa29I, BshVI, BspDI, BspXI, BsuTUI, ClaI BsaJI, BssECI BstF5I, BtsCI, [FokI] BsaBI, Bse8I AfiI, Bsc4I, BslI Bse3DI, BsrDI [BspCNI] Bse1I, BsrI, BsrSI BssHII BaeGI, BstSLI BbvI, BstV1I BstZI, EagI, EclXI [GsaI] (continued on next page)
FastDigest Bme1580I (BseSI) FastDigest BseXI, FastDigest BbvI (Lsp1109I) FastDigest EagI (Eco52I) [FastDigest PspFI] (CCCAGC(-1/-5))
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Enzyme BsgI Bsh1236I Bsh1285I BshFI BshNI BshTI BshVI BsiEI BsiHKAI BsiHKCI BsiSI BsiWI BslFI BslI BsmAI BsmBI BsmFI BsmI BsnI Bso31I BsoBI Bsp119I Bsp120I Bsp1286I Bsp1407I Bsp143I Bsp13I Bsp1720I Bsp19I Bsp68I BspACI BspANI BspCNI BspDI BspEI BspFNI BspHI BspLI BspMAI BspMI BspOI BspPI BspQI BspT104I BspT107I BspTI BspTNI BspXI BsrBI BsrDI BsrFI BsrGI BsrI BsrSI BssAI BssECI BssHII BssKI
Specificity 53 GTGCAG(16/14) CGCG CGRYCG GGCC GGYRCC ACCGGT ATCGAT CGRYCG GWGCWC CYCGRG CCGG CGTACG GGGAC(10/14) CCNNNNNNNGG GTCTC(1/5) CGTCTC(1/5) GGGAC(10/14) GAATGC(1/-1) GGCC GGTCTC(1/5) CYCGRG TTCGAA GGGCCC GDGCHC TGTACA GATC TCCGGA GCTNAGC CCATGG TCGCGA CCGC(-3/-1) GGCC CTCAG(9/7) ATCGAT TCCGGA CGCG TCATGA GGNNCC CTGCAG ACCTGC(4/8) GCTAGC GGATC(4/5) GCTCTTC(1/4) TTCGAA GGYRCC CTTAAG GGTCTC(1/5) ATCGAT CCGCTC(-3/-3) GCAATG(2/0) RCCGGY TGTACA ACTGG(1/-1) ACTGG(1/-1) RCCGGY CCNNGG GCGCGC CCNGG
FastDigest enzyme FastDigest Bsh1236I FastDigest BsiEI (Bsh1285I) FastDigest HaeIII (BsuRI) FastDigest BanI (BshNI) FastDigest AgeI (BshTI) FastDigest ClaI (Bsu15I) FastDigest BsiEI (Bsh1285I)m FastDigest Alw21I FastDigest AvaI (Eco88I) FastDigest HpaIIm, FastDigest MspI FastDigest BsiWI (Pfl23II) FastDigest BsmFI (FaqI) FastDigest BslI (BseLI) FastDigest Alw26Im FastDigest BsmBI (Esp3I) FastDigest BsmFI (FaqI) FastDigest Mva1269I FastDigest HaeIII (BsuRI) FastDigest Eco31I FastDigest AvaI (Eco88I)m FastDigest Bsp119I [FastDigest ApaI](GGGCCC), FastDigest Bsp120I FastDigest Bsp1286I (SduI) FastDigest Bsp1407I FastDigest Sau3AI (Bsp143I), FastDigest MboIm FastDigest Kpn2I FastDigest BlpI (Bpu1102I) FastDigest NcoI FastDigest NruI (RruI) FastDigest AciI (SsiI) FastDigest HaeIII (BsuRI) [FastDigest BspCNI (BseMII)] (CTCAG(10/8)) FastDigest ClaI (Bsu15I) FastDigest Kpn2I FastDigest Bsh1236I FastDigest BspHI (PagI)m FastDigest NlaIV (BspLI) FastDigest PstI FastDigest BspMI (BveI) FastDigest BmtI (BspOI), [FastDigest NheI](GCTAGC)
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Page Conventional enzyme 25 26 40 19 13 32 26 15 17 42 48 26 27 26 15 27 27 49 40 36 17 27 16 28 28 28 60 44 44 21 50 52 12 40 29 32 44 25 29 51 56 29 22 50 59 27 19 13 36 32 30 30 30 28 25 25 30 24 31 61 Bsh1236I (BstUI) Bsh1285I (BsiEI) BsuRI (HaeIII) BshNI (BanI) BshTI (AgeI) Bsu15I (ClaI) Bsh1285I (BsiEI)m Alw21I (BsiHKAI) Eco88I (AvaI) HpaIIm, MspI (HpaII) Pfl23II (BsiWI) FaqI (BsmFI) BseLI (BslI) Alw26I (BsmAI)m Esp3I (BsmBI) FaqI (BsmFI) Mva1269I (BsmI) BsuRI (HaeIII) Eco31I (BsaI) Eco88I (AvaI)m Bsp119I (BstBI) [ApaI](GGGCCC), Bsp120I (PspOMI) SduI (Bsp1286I) Bsp1407I (BsrGI) Bsp143I (Sau3AI), MboIm Kpn2I (BspEI) Bpu1102I (BlpI) NcoI Bsp68I (NruI) SsiI (AciI) BsuRI (HaeIII) [BseMII (BspCNI)](CTCAG(10/8)) Bsu15I (ClaI) Kpn2I (BspEI) Bsh1236I (BstUI) PagI (BspHI)m BspLI (NlaIV) PstI BveI (BspMI) BspOI (BmtI), [NheI](GCTAGC) BspPI (AlwI) LguI (SapI) Bsp119I (BstBI) BshNI (BanI) BspTI (AflII) Eco31I (BsaI)
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Page 97 97 103 97 98 103 97 84 113 122 129 137 118 95 85 117 118 131 103 110 113 99 85 99 145 100 99 127 124 93 131 98 149 103 95 103 124 97 135 95 140 104 100 132 101 125 99 97 101 110 103 126 95 105 100 96 96 105 94 135 91
Commercially available isoschizomers from other vendors AccII, BspFNI, BstFNI, BstUI, MvnI BsiEIm, BstMCI BsnI, BspANI, HaeIII, PhoI AccB1I, BanI, BspT107I AgeI, AsiGI, CspAIm, PinAI BanIII, Bsa29I, BseCI, BspDI, BspXI, BsuTUI, ClaI BsiEI, BstMCI Bbv12I Ama87I, AvaI, [BmeT110I], BsoBI HapIIm BsiWI, PspLI BsmFI AfiI, Bsc4I, BslI BstMAI BsmBI BslFI, BsmFI BsaMI, PctI BshFI, BspANI, HaeIII, PhoI BsaI, BspTNI Ama87I, AvaIm, [BmeT110I], BsiHKCI AsuIIm, Bpu14I, BspT104I, BstBI, Csp45I, NspV, SfuIm PspOMI Bsp1286I, MhlI BsrGI, BstAUI BfuCI, BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm, Sau3AIm AccIII, Aor13HI, BseAI, BspEI, MroI BlpI, CelII BtuMI, NruI AciI BshFI, BsnI, HaeIII, PhoI BspCNI BanIII, Bsa29I, BseCI, BshVI, BspXI, BsuTUI, ClaI AccIIIm, Aor13HI, BseAIm, Bsp13I, MroIm AccII, BstFNI, BstUI, MvnI BspHI, CciI, RcaI BmiI, NlaIVm, PspN4I Acc36I, BfuAI, BspMI [AsuNHI], BmtI AclWI, AlwI PciSI, SapI AsuIIm, Bpu14I, BstBI, Csp45I, NspV, SfuIm AccB1I, BanI AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464I BsaI, Bso31I BanIII, Bsa29I, BseCI, BshVI, BspDI, BsuTUI, ClaI AccBSI, BsrBI Bse3DI, BsrDI Bse118I, BsrFI, BssAIm BstAUI Bse1I, BsrSI Bse1I, BsrI Bse118I, BsrFIm BsaJI BsePI, BssHII [BmrFI], BstSCI, [MspR9I], [ScrFI], StyD4I (continued on next page)
FastDigest SapI (LguI) FastDigest Bsp119I FastDigest BanI (BshNI) FastDigest AflII (BspTI) FastDigest Eco31I FastDigest ClaI (Bsu15I) FastDigest BsrBI (MbiI) FastDigest BsrDI (BseMI) FastDigest BsrFI (Cfr10I) FastDigest Bsp1407I FastDigest BseNI FastDigest BseNI FastDigest BsrFI (Cfr10I)m FastDigest BsaJI (BseDI) FastDigest BssHII (PteI) [FastDigest ScrFI (Bme1390I)](CCNGG)
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Bsu15I (ClaI) MbiI (BsrBI) BseMI (BsrDI) Cfr10I (BsrFI) Bsp1407I (BsrGI) BseNI (BsrI) BseNI (BsrI) Cfr10I (BsrFI)m BseDI (BsaJI) PauI (BssHII) [Bme1390I (ScrFI)](CCNGG)
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Enzyme BssMI BssNAI BssNI BssSI BssT1I Bst1107I Bst2BI Bst2UI Bst4CI Bst6I Bst98I BstACI BstAFI BstAPI BstAUI BstBAI BstBI BstC8I BstDEI BstDSI BstEII BstENI BstF5I BstFNI BstH2I BstHHI BstKTI BstMAI BstMBI BstMCI BstMWI BstNI BstNSI BstOI BstPAI BstPI BstSCI BstSFI BstSLI BstSNI BstUI BstV1I BstV2I BstX2I BstXI BstYI BstZ17I BstZI Bsu15I Bsu36I BsuRI BsuTUI BtgI BtgZI BtrI Specificity 53 GATC GTATAC GRCGYC CACGAG(-5/-1) CCWWGG GTATAC CACGAG(-5/-1) CCWGG ACNGT CTCTTC(1/4) CTTAAG GRCGYC CTTAAG GCANNNNNTGC TGTACA YACGTR TTCGAA GCNNGC CTNAG CCRYGG GGTNACC CCTNNNNNAGG GGATG(2/0) CGCG RGCGCY GCGC GATC GTCTC(1/5) GATC CGRYCG GCNNNNNNNGC CCWGG RCATGY CCWGG GACNNNNGTC GGTNACC CCNGG CTRYAG GKGCMC TACGTA CGCG GCAGC(8/12) GAAGAC(2/6) RGATCY CCANNNNNNTGG RGATCY GTATAC CGGCCG ATCGAT CCTNAGG GGCC ATCGAT CCRYGG GCGATG(10/14) CACGTC(-3/-3) FastDigest enzyme FastDigest Sau3AI (Bsp143I), FastDigest MboI FastDigest BstZ17I (Bst1107I) FastDigest BsaHI (Hin1I) FastDigest StyI (Eco130I) FastDigest BstZ17I (Bst1107I) FastDigest MvaI FastDigest TaaI FastDigest EarI (Eam1104I) FastDigest AflII (BspTI) FastDigest BsaHI (Hin1I) FastDigest AflII (BspTI)
Page Conventional enzyme 60 44 31 24 65 31 48 65 35 13 24 13 28 23 27 33 36 36 24 38 25 41 40 42 60 44 15 60 44 26 43 48 53 48 55 36 61 62 21 63 25 19 25 19 56 31 56 31 34 32 32 40 32 Bsp143I (Sau3AI), MboI Bst1107I (BstZ17I) Hin1I (BsaHI) BauI (BssSI) Eco130I (StyI) Bst1107I (BstZ17I) BauI (BssSI) [EcoRIIm](CCWGG), MvaI (BstNI) TaaI (HpyCH4III) Eam1104I (EarI) BspTI (AflII) Hin1I (BsaHI) BspTI (AflII) Bsp1407I (BsrGI) Ppu21I (BsaAI) Bsp119I (BstBI) HpyF3I (DdeI) Eco91I (BstEII) XagI (EcoNI) BseGI (BtsCI) Bsh1236I (BstUI) HhaI, [Hin6I (HinP1I)](GCGC) [Bsp143I (Sau3AI)m](GATC), [MboI](GATC) Alw26I (BsmAI) Bsp143I (Sau3AI), MboI Bsh1285I (BsiEI) HpyF10VI (MwoI) [EcoRIIm](CCWGG), MvaI (BstNI) XceI (NspI) [EcoRIIm](CCWGG), MvaI (BstNI) BoxI (PshAI) Eco91I (BstEII) [Bme1390I (ScrFI)](CCNGG) BfmI (SfcI) BseSI (Bme1580I) Eco105I (SnaBI) Bsh1236I (BstUI) Lsp1109I (BbvI), BseXI (BbvI) BpiI (BbsI) PsuI (BstYI) BstXI PsuI (BstYI) Bst1107I (BstZ17I) Eco52I (EagI) Bsu15I (ClaI) Eco81I (Bsu36I) BsuRI (HaeIII) Bsu15I (ClaI)
Page 99 127 102 120 87 114 102 87 116 130 150 109 101 120 101 100 138 99 123 114 155 94 97 120 121 99 127 85 99 127 97 124 116 130 156 116 130 91 114 91 89 96 114 97 125 96 92 140 102 140 102 111 103 113 103 103
Commercially available isoschizomers from other vendors BfuCI, [BstKTI], BstMBI, DpnII, Kzo9I, NdeII, Sau3AI BstZ17I AcyI, BsaHI, BstACI, Hsp92I Bst2BI EcoT14I, ErhI, StyI BssNAI, BstZ17I BssSI [AjnI], BseBI, BstNI, BstOI, [Psp6I], [PspGIm] HpyCH4III EarI AflII, BfrI, BstAFI, MspCI, Vha464I AcyI, BsaHI, BssNI, Hsp92I AflII, BfrI, Bst98I, MspCI, Vha464I BsrGI BsaAI AsuIIm, Bpu14I, BspT104I, Csp45I, NspVm, SfuIm Cac8I DdeI BtgI BstPI, EcoO65I, PspEI EcoNI BtsCI, [FokI] AccII, BspFNI, BstUI, MvnI HaeII AspLEI, CfoI, [HinP1I], [HspAI] [BfuCIm], [BssMI], [BstMBI], [DpnII], [Kzo9Im], [NdeIIm], [Sau3AIm] BsmAI BfuCI, BssMI, [BstKTI], DpnII, Kzo9I, NdeII, Sau3AI BsiEI MwoI [AjnI], BseBI, Bst2UI, BstOI, [Psp6I], [PspGIm] NspI [AjnI], BseBI, Bst2UI, BstNI, [Psp6I], [PspGIm] PshAI BstEII, EcoO65I, PspEI [BmrFI], BssKI, [MspR9I], [ScrFI], StyD4I SfcI BaeGI SnaBI AccII, BspFNI, BstFNI, MvnI BbvI BbsI, BpuAI BstYI, MflI, XhoII BstX2I, MflIm, XhoII BssNAI, BstZ17I BseX3I, EagI, EclXI BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI AxyI, Bse21I, Bsu36I BshFI, BsnI, BspANI, HaeIII, PhoI BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, ClaI BstDSI BmgBI (continued on next page)
FastDigest Bsp1407I FastDigest BsaAI (Ppu21I) FastDigest Bsp119I FastDigest DdeI (HpyF3I) FastDigest Eco91I FastDigest EcoNI (XagI) FastDigest BseGI, [FastDigest FokI](GGATG(9/13)) FastDigest Bsh1236I FastDigest HaeII (BfoI) FastDigest HhaI, [FastDigest HinP1I (Hin6I)](GCGC) [FastDigest Sau3AI (Bsp143I)m](GATC), [FastDigest MboI](GATC) FastDigest Alw26I FastDigest Sau3AI (Bsp143I), FastDigest MboI FastDigest BsiEI (Bsh1285I) FastDigest HpyF10VI FastDigest MvaI FastDigest NspI (XceI)
FastDigest MvaI FastDigest PshAI (BoxI) FastDigest Eco91I [FastDigest ScrFI (Bme1390I)](CCNGG) FastDigest SfcI (BfmI) FastDigest Bme1580I (BseSI) FastDigest SnaBI (Eco105I) FastDigest Bsh1236I FastDigest BbvI (Lsp1109I), FastDigest BseXI FastDigest BbsI (BpiI) FastDigest PsuI FastDigest BstXI FastDigest PsuI FastDigest BstZ17I (Bst1107I) FastDigest EagI (Eco52I) FastDigest ClaI (Bsu15I) FastDigest Bsu36I (Eco81I) FastDigest HaeIII (BsuRI) FastDigest ClaI (Bsu15I)
AjiI (BmgBI)
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Enzyme BtsCI BtsI BtuMI BveI Cac8I CaiI CciI CciNI CelII CfoI Cfr10I Cfr13I Cfr42I Cfr9I CfrI ClaI CpoI CseI Csp45I Csp6I CspAI CspCI CspI CviAII CviJI CviKI-1 CviQI DdeI DinI DpnI DpnII DraI DraII DraIII DrdI DriI DseDI EaeI EagI Eam1104I Eam1105I EarI EciI Ecl136II EclHKI EclXI Eco105I Eco130I Eco147I Eco24I Eco31I Eco32I Eco47I Eco47III Eco52I Eco53kI Eco57I
Specificity 53 GGATG(2/0) GCAGTG(2/0) TCGCGA ACCTGC(4/8) GCNNGC CAGNNNCTG TCATGA GCGGCCGC GCTNAGC GCGC RCCGGY GGNCC CCGCGG CCCGGG YGGCCR ATCGAT CGGWCCG GACGC(5/10) TTCGAA GTAC ACCGGT (11/13)CAA(N)5GTGG(12/10) CGGWCCG CATG RGCY RGCY GTAC CTNAG GGCGCC Gm6ATC GATC TTTAAA RGGNCCY CACNNNGTG GACNNNNNNGTC GACNNNNNGTC GACNNNNNNGTC YGGCCR CGGCCG CTCTTC(1/4) GACNNNNNGTC CTCTTC(1/4) GGCGGA(11/9) GAGCTC GACNNNNNGTC CGGCCG TACGTA CCWWGG AGGCCT GRGCYC GGTCTC(1/5) GATATC GGWCC AGCGCT CGGCCG GAGCTC CTGAAG(16/14)
FastDigest enzyme FastDigest BseGI, [FastDigest FokI](GGATG(9/13))
Page Conventional enzyme 24 38 52 29 15 29 52 21 40 42 30 60 63 BseGI (BtsCI) Bsp68I (NruI) BveI (BspMI) CaiI (AlwNI) PagI (BspHI) NotI Bpu1102I (BlpI) HhaI, [Hin6I (HinP1I)](GCGC) Cfr10I (BsrFI) Cfr13I (Sau96I) Cfr42I (SacII) Cfr9I (XmaI), [SmaIm](CCCGGG) CfrI (EaeI) Bsu15I (ClaI) CpoI (RsrII) CseI (HgaI) Bsp119I (BstBI) Csp6I (CviQI), [RsaIm](GTAC) BshTI (AgeI)m CpoI (RsrII) [Hin1II (NlaIII)](CATG)
Page 94 98 104 104 135 133 93 120 121 105 106 106 105 147 105 103 106 107 99 107 141 98 106 120
Commercially available isoschizomers from other vendors BstF5I, [FokI] NruI Acc36I, BfuAI, BspMI BstC8I AlwNI BspHI, RcaI BlpI, Bsp1720I AspLEI, BstHHI, [HinP1I], [HspAI] Bse118I, BsrFI, BssAIm AspS9I, [BmgT120I], PspPI, Sau96I KspI, SacII, Sfr303I, SgrBI, SstII TspMIm, XmaCI, XmaIm AcoI, EaeI BanIII, Bsa29I, BseCI, BshVI, BspDI, BspXI, BsuTUI, ClaI CspI, Rsr2I, RsrII HgaI AsuII, Bpu14I, BspT104I, BstBI, NspVm, SfuI [AfaIm], CviQI, RsaNI AgeIm, AsiGI, PinAI Rsr2I, RsrII [FaeI], [FatIm][Hsp92II], [NlaIII] CviKI-1 CviJI [AfaIm], RsaNI BstDEI, DdeI [BbeI], EgeI, [KasI], [Mly113I], [NarI], SfoI MalI BfuCIm, BssMI, [BstKTIm], BstMBI, Kzo9Im, NdeII, Sau3AIm
FastDigest NruI (RruI) FastDigest BspMI (BveI) FastDigest AlwNI (CaiI) FastDigest BspHI (PagI) FastDigest NotI FastDigest BlpI (Bpu1102I) FastDigest HhaI, [FastDigest HinP1I (Hin6I)](GCGC) FastDigest BsrFI (Cfr10I) FastDigest Sau96I (Cfr13I) [FastDigest SmaIm](CCCGGG)
FastDigest ClaI (Bsu15I) FastDigest RsrII (CpoI) FastDigest HgaI (CseI) FastDigest Bsp119I FastDigest Csp6I, [FastDigest RsaIm](GTAC) FastDigest AgeI (BshTI)m
32 58 40 27 32 58 13 58 51
FastDigest RsrII (CpoI) [FastDigest NlaIII (Hin1II)](CATG)
FastDigest Csp6I, [FastDigest RsaIm](GTAC) FastDigest DdeI (HpyF3I) FastDigest EheI
32 58 33 117 33 60 44 33 37 34 34 35 34 34 35 35 35 35 58 35 34 63 65 64 36 37 18 13 34 35 58 12
FastDigest DpnI FastDigest Sau3AI (Bsp143I)m, FastDigest MboIm FastDigest DraI FastDigest EcoO109I FastDigest DraIII (AdeI) FastDigest DrdI (AasI) FastDigest Eam1105I FastDigest DrdI (AasI) FastDigest EagI (Eco52I) FastDigest EarI (Eam1104I) FastDigest Eam1105I FastDigest EarI (Eam1104I) FastDigest Ecl136II, [FastDigest SacIm](GAGCTC) FastDigest Eam1105I FastDigest EagI (Eco52I) FastDigest SnaBI (Eco105I) FastDigest StyI (Eco130I) FastDigest StuI (Eco147I) FastDigest Eco31I FastDigest EcoRV (Eco32I) FastDigest AvaII (Eco47I) FastDigest AfeI (Eco47III) FastDigest EagI (Eco52I) FastDigest Ecl136IIm, [FastDigest SacIm](GAGCTC) FastDigest AcuI (Eco57I)
Csp6I (CviQI), [RsaIm](GTAC) HpyF3I (DdeI) EheI (SfoI), [SspDI (KasI)](GGCGCC) DpnI [Bsp143I (Sau3AI)m], MboIm DraI EcoO109I (DraII) AdeI (DraIII) AasI (DrdI) Eam1105I (AhdI) AasI (DrdI) CfrI (EaeI) Eco52I (EagI) Eam1104I (EarI) Eam1105I (AhdI) Eam1104I (EarI) Ecl136II (EcoICRI), [SacIm](GAGCTC) Eam1105I (AhdI) Eco52I (EagI) Eco105I (SnaBI) Eco130I (StyI) Eco147I (StuI) Eco24I (BanII) Eco31I (BsaI) Eco32I (EcoRV) Eco47I (AvaII) Eco47III (AfeI) Eco52I (EagI) Ecl136II (EcoICRI)m, [SacIm](GAGCTC) Eco57I (AcuI)
107 141 123 117 150 108 99 127 108 115 82 80 109 80 105 111 109 109 109 109 142 109 111 114 114 115 110 110 110 111 111 111 109 142 112
DraIII DrdI, DseDI AhdI, AspEI, BmeRI, EclHKI DrdI AcoI BseX3I, BstZI, EagI, EclXI Bst6I, EarI AhdI, AspEI, BmeRI, DriI, EclHKI Bst6I, EarI Eco53kIm, EcoICRI, [Psp124BI], [SstIm] AhdI, AspEI, BmeRI, DriI BseX3I, BstZI, EagI BstSNI, SnaBI BssT1I, EcoT14I, ErhI, StyI AatI, PceI, SseBI, StuI BanII, EcoT38I, FriOI BsaI, Bso31I, BspTNI EcoRV AvaII, Bme18I, SinI, VpaK11BI AfeI, Aor51HI BseX3I, BstZI, EagI, EclXI EcoICRI, [Psp124BI], [SstI] AcuI (continued on next page)
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Enzyme Eco57MI Eco72I Eco81I Eco88I Eco91I EcoICRI EcoNI EcoO109I EcoO65I EcoP15I EcoRI EcoRII EcoRV EcoT14I EcoT22I EcoT38I EgeI EheI ErhI Esp3I FaeI FaiI FaqI FalI FatI FauI FauNDI FbaI FblI Fnu4HI FokI FriOI FseI Fsp4HI FspAI FspBI FspI GlaI GluI GsaI GsuI HaeII HaeIII HapII HgaI HhaI Hin1I Hin1II Hin4I Hin6I HincII HindII HindIII HinfI HinP1I HpaI HpaII Specificity 53 CTGRAG(16/14) CACGTG CCTNAGG CYCGRG GGTNACC GAGCTC CCTNNNNNAGG RGGNCCY GGTNACC CAGCAG(25/27) GAATTC CCWGG GATATC CCWWGG ATGCAT GRGCYC GGCGCC GGCGCC CCWWGG CGTCTC(1/5) CATG YATR GGGAC(10/14) (8/13)AAG(N)5CTT(13/8) CATG CCCGC(4/6) CATATG TGATCA GTMKAC GCNGC GGATG(9/13) GRGCYC GGCCGGCC GCNGC RTGCGCAY CTAG TGCGCA GCGC GCNGC CCCAGC(-1/-5) CTGGAG(16/14) RGCGCY GGCC CCGG GACGC(5/10) GCGC GRCGYC CATG (8/13)GAY(N)5VTC(13/8) GCGC GTYRAC GTYRAC AAGCTT GANTC GCGC GTTAAC CCGG FastDigest enzyme FastDigest PmlI (Eco72I) FastDigest Bsu36I (Eco81I)
Page Conventional enzyme 54 32 17 36 35 58 36 37 36 37 48 37 65 52 38 38 65 27 51 27 51 50 20 11 38 24 38 Eco57MI Eco72I (PmlI) Eco81I (Bsu36I) Eco88I (AvaI) Eco91I (BstEII) Ecl136II (EcoICRI), [SacI](GAGCTC) XagI (EcoNI) EcoO109I (DraII) Eco91I (BstEII) EcoRI EcoRII, [MvaI (BstNI)m](CCWGG) Eco32I (EcoRV) Eco130I (StyI) Mph1103I (NsiI) Eco24I (BanII) EheI (SfoI), [SspDI (KasI)](GGCGCC) EheI (SfoI), [SspDI (KasI)](GGCGCC) Eco130I (StyI) Esp3I (BsmBI) Hin1II (NlaIII) FaqI (BsmFI) [Hin1II (NlaIII)](CATG) SmuI (FauI) NdeI BclI XmiI (AccI) SatI (Fnu4HI) [BseGI (BtsCI)](GGATG(2/0)) Eco24I (BanII)
Page 112 113 113 113 114 109 142 155 115 114 116 116 130 110 114 129 110 117 150 117 150 114 117 120 118 120 148 132 87 157 143 94 110 143 118 119 133
Commercially available isoschizomers from other vendors AcvI, BbrPI, PmaCI, PmlI, PspCI AxyI, Bse21I, Bsu36I Ama87I, AvaIm, [BmeT110Im], BsiHKCI, BsoBIm BstEII, BstPI, EcoO65I, PspEI Eco53kI, [Psp124BI], [SstI] BstENI, EcoNI DraII BstEII, BstPI, PspEI
FastDigest AvaI (Eco88I) FastDigest Eco91I FastDigest Ecl136II, [FastDigest SacI](GAGCTC) FastDigest EcoNI (XagI) FastDigest EcoO109I FastDigest Eco91I
FastDigest EcoRI [FastDigest MvaI ](CCWGG)

m
FastDigest EcoRV (Eco32I) FastDigest StyI (Eco130I) FastDigest NsiI (Mph1103I) FastDigest EheI FastDigest EheI FastDigest StyI (Eco130I) FastDigest BsmBI (Esp3I) FastDigest NlaIII (Hin1II)
AjnIm, [BseBI], [Bst2UIm], [BstNIm], [BstOIm], Psp6I, PspGI EcoRV BssT1I, ErhI, StyI NsiIm, Zsp2I BanII, FriOI [BbeI], DinI, [KasI], [Mly113I], [NarI], SfoI [BbeI], DinI, EgeI, [KasI], [Mly113I], [NarI], SfoIm BssT1I, EcoT14I, StyI BsmBI [CviAII], [FatI], Hsp92II, NlaIII BslFI, BsmFI [CviAIIm], [FaeI], [Hsp92II], [NlaIIIm]
FastDigest BsmFI (FaqI) [FastDigest NlaIII (Hin1II)](CATG) FastDigest NdeI FastDigest BclI FastDigest AccI (XmiI) FastDigest Fnu4HI (SatI) [FastDigest BseGI](GGATG(2/0)), FastDigest FokI
Ksp22I AccI Fnu4HI, Fsp4HI, ItaIm [BstF5I], [BtsCI], FokI BanII, EcoT38I RigI Fnu4HI, ItaIm BfaI, MaeI, XspI Acc16I, AviII, FspI BisI, [BlsI] [BseYI] BpmIm BstH2I, HaeII BshFI, BsnI, BspANI, HaeIII, PhoI BsiSIm HgaI AspLEI, BstHHI, CfoI, [HinP1I], [HspAI] AcyI, BsaHIm, BssNI, BstACI, Hsp92I [CviAII], FaeI, [FatI], Hsp92II, NlaIII [AspLEI], [BstHHI], [CfoI], HinP1I, HspAI HindII
FastDigest Fnu4HI (SatI) FastDigest FspAI FastDigest BfaI (FspBI) FastDigest FspI (NsbI)
38 39 20 39
SatI (Fnu4HI) FspAI FspBI (BfaI) NsbI (FspI)
FastDigest PspFI FastDigest BpmI (GsuI) FastDigest HaeII (BfoI) FastDigest HaeIII (BsuRI) FastDigest HpaII, FastDigest MspIm FastDigest HgaI (CseI) FastDigest HhaI, [FastDigest HinP1I (Hin6I)](GCGC) FastDigest BsaHI (Hin1I) FastDigest NlaIII (Hin1II) [FastDigest HhaI](GCGC), FastDigest HinP1I (Hin6I) FastDigest HincII FastDigest HincII FastDigest HindIII FastDigest HinfI [FastDigest HhaI](GCGC), FastDigest HinP1I (Hin6I) FastDigest HpaI (KspAI)m FastDigest HpaII, FastDigest MspIm
56 22 39 40 42 48 40 40 42 24 51 40 42 41 41 41 41 40 42 42 42 48
GsuI (BpmI) BsuRI (HaeIII) HpaII, MspI (HpaII)m CseI (HgaI) HhaI, [Hin6I (HinP1I)](GCGC) Hin1I (BsaHI) Hin1II (NlaIII) Hin4I [HhaI](GCGC), Hin6I (HinP1I) HincII (HindII) HincII (HindII) HindIII HinfI [HhaI](GCGC), Hin6I (HinP1I) KspAI (HpaI)m HpaII, MspI (HpaII)m
119 103 122 129 107 120 121 120 120 121 120 121 121 121 122 122 120 121 125 122 129
[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI HpaI BsiSIm, HapII (continued on next page)
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Enzyme HphI Hpy166II Hpy188I Hpy188III Hpy8I Hpy99I HpyAV HpyCH4III HpyCH4IV HpyCH4V HpyF10VI HpyF3I Hsp92I Hsp92II HspAI ItaI KasI Kpn2I KpnI Ksp22I KspAI KspI Kzo9I LguI Lsp1109I LweI MabI MaeI MaeII MaeIII MalI MauBI MbiI MboI MboII MfeI MflI MhlI MlsI MluI MluNI Mly113I MlyI MmeI MnlI Mph1103I MreI MroI MroNI MroXI MscI MseI MslI Msp20I MspA1I MspCI MspI MspR9I
Specificity 53 GGTGA(8/7) GTNNAC TCNGA TCNNGA GTNNAC CGWCG CCTTC(6/5) ACNGT ACGT TGCA GCNNNNNNNGC CTNAG GRCGYC CATG GCGC GCNGC GGCGCC TCCGGA GGTACC TGATCA GTTAAC CCGCGG GATC GCTCTTC(1/4) GCAGC(8/12) GCATC(5/9) ACCWGGT CTAG ACGT GTNAC GATC CGCGCGCG CCGCTC(-3/-3) GATC GAAGA(8/7) CAATTG RGATCY GDGCHC TGGCCA ACGCGT TGGCCA GGCGCC GAGTC(5/5) TCCRAC(20/18) CCTC(7/6) ATGCAT CGCCGGCG TCCGGA GCCGGC GAANNNNTTC TGGCCA TTAA CAYNNNNRTG TGGCCA CMGCKG CTTAAG CCGG CCNGG
FastDigest enzyme FastDigest Hpy8I
Page Conventional enzyme 43 HphI Hpy8I (MjaIV)
Page 123 123
Commercially available isoschizomers from other vendors AsuHPI
FastDigest Hpy8I
43
Hpy8I (MjaIV)
123
Hpy166II
FastDigest TaaI [FastDigest TaiIm](ACGT) FastDigest HpyF10VI FastDigest DdeI (HpyF3I) FastDigest BsaHI (Hin1I) FastDigest NlaIII (Hin1II) [FastDigest HhaI](GCGC), FastDigest HinP1I (Hin6I) FastDigest Fnu4HI (SatI)m [FastDigest EheI](GGCGCC) FastDigest Kpn2I
65 66 43 33 24 51 40 42 38 38 44
TaaI (HpyCH4III) [TaiI (MaeII)m](ACGT) HpyF10VI (MwoI) HpyF3I (DdeI) Hin1I (BsaHI) Hin1II (NlaIII) [HhaI](GCGC), Hin6I (HinP1I) SatI (Fnu4HI)m [EheI (SfoI)](GGCGCC), SspDI (KasI) Kpn2I (BspEI) [Acc65I (Asp718I) ](GGTACC), KpnI BclI KspAI (HpaI) Cfr42I (SacII) Bsp143I (Sau3AI), MboIm LguI (SapI) Lsp1109I (BbvI), BseXI (BbvI) LweI (SfaNI)
m
150 150 124 123 120 120 120 121 143 117 150 124 81 124 87 125 106 99 127 125 125 96 126 119 150 108 126 126 99 127 127 130 140 145 128 128 128 117 150 144 128 129 129 124 136 136 128 152 142 128 101 122 129 91
Bst4CI MaeII BstMWI, MwoIm BstDEI, DdeI AcyI, BsaHI, BssNI, BstACI [CviAII], FaeI, [FatI], NlaIII [AspLEI], [BstHHI], [CfoI], HinP1I Fnu4HIm, Fsp4HIm [BbeI], [DinI], [EgeI], [Mly113I], [NarI], [SfoIm] AccIIIm, Aor13HI, BseAIm, Bsp13I, BspEIm, MroI [Asp718Im] FbaI HpaI SacII, Sfr303I, SgrBI, SstII BfuCI, BssMI, [BstKTIm], BstMBI, DpnIIm, NdeIIm, Sau3AI BspQI, PciSI, SapI BbvI, BstV1I SfaNI SexAIm BfaI, XspI HpyCH4IV
[FastDigest Acc65I ](GGTACC), FastDigest KpnI FastDigest BclI FastDigest HpaI (KspAI)
m
11 43 20 42 60 44 59 19 25 62 61 20 66 33 44 30 60 44 45 45 56 28 47 45 47 38 46 46 52 46 44 49 53 47 47 67 47 47 13 42 48 61
FastDigest Sau3AI (Bsp143I), FastDigest MboIm FastDigest SapI (LguI) FastDigest BbvI (Lsp1109I), FastDigest BseXI FastDigest SfaNI (BmsI) FastDigest SexAI (CsiI) FastDigest BfaI (FspBI) [FastDigest TaiI](ACGT) FastDigest DpnI FastDigest MauBI FastDigest BsrBI (MbiI) FastDigest Sau3AI (Bsp143I)m, FastDigest MboI FastDigest MboII FastDigest MfeI (MunI) FastDigest PsuIm FastDigest Bsp1286I (SduI) FastDigest MscI (MlsI) FastDigest MluI FastDigest MscI (MlsI) [FastDigest EheI](GGCGCC)
FspBI (BfaI) [TaiI (MaeII)](ACGT) DpnI MauBI MbiI (BsrBI) Bsp143I (Sau3AI)m, MboI MboII MunI (MfeI) PsuI (BstYI)m SduI (Bsp1286I) MlsI (MscI) MluI MlsI (MscI) [EheI (SfoI)](GGCGCC), [SspDI (KasI)](GGCGCC) SchI (MlyI) MnlI Mph1103I (NsiI) MreI (Sse232I) Kpn2I (BspEI) [PdiI (NaeI)](GCCGGC) PdmI (XmnI) MlsI (MscI) Tru1I (MseI) RseI (MslI) MlsI (MscI) BspTI (AlfII) HpaIIm, MspI (HpaII) Bme1390I (ScrFI)
AccBSI, BsrBIm BfuCIm, BssMI, [BstKTI], BstMBI, DpnIIm, Kzo9Im, NdeIIm, Sau3AIm MfeI BstX2I, BstYIm, XhoIIm Bsp1286I BalI, MluNI, MscI, Msp20I BalI, MscI, Msp20I [BbeI], [DinI], [EgeI], [KasI], NarI, [SfoI] MlyI, [PleIm], [PpsI]
FastDigest MlyI (SchI) FastDigest MnlI FastDigest NsiI (Mph1103I) FastDigest MreI FastDigest Kpn2I [FastDigest NaeI (PdiI)](GCCGGC) FastDigest PdmI FastDigest MscI (MlsI) FastDigest MseI (SaqAI), FastDigest Tru1I FastDigest MslI (RseI) FastDigest MscI (MlsI) FastDigest AflII (BspTI) FastDigest HpaIIm, FastDigest MspI FastDigest ScrFI (Bme1390I)
EcoT22I, NsiI, Zsp2I AccIIIm, Aor13HI, BseAIm, Bsp13I, BspEIm [NaeI], NgoMIV Asp700I, XmnI BalI, MscI, MluNI, Msp20I MseI, Tru9I MslI, SmiMI BalI, MluNI, MscI AflII, BfrI, Bst98I, BstAFI, Vha464I BsiSI, HapIIm BmrFI, [BssKI], [BstSCI], ScrFI, [StyD4I] (continued on next page)
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Enzyme MssI MunI Mva1269I MvaI MvnI MvrI MwoI NaeI NarI NciI NcoI NdeI NdeII NgoMIV NheI NlaIII NlaIV NmeAIII NmuCI NotI NruI NsbI NsiI NspI NspV OliI PacI PaeI PaeR7I PagI PalAI PasI PauI PceI PciI PciSI PctI PdiI PdmI PfeI Pfl23II PflFI PflMI PfoI PhoI PinAI Ple19I PleI PmaCI PmeI PmlI PpiI PpsI Ppu21I PpuMI PscI PshAI PshBI PsiI Psp124BI Psp1406I Specificity 53 GTTTAAAC CAATTG GAATGC(1/-1) CCWGG CGCG CGATCG GCNNNNNNNGC GCCGGC GGCGCC CCSGG CCATGG CATATG GATC GCCGGC GCTAGC CATG GGNNCC GCCGAG(21/19) GTSAC GCGGCCGC TCGCGA TGCGCA ATGCAT RCATGY TTCGAA CACNNNNGTG TTAATTAA GCATGC CTCGAG TCATGA GGCGCGCC CCCWGGG GCGCGC AGGCCT ACATGT GCTCTTC(1/4) GAATGC(1/-1) GCCGGC GAANNNNTTC GAWTC GGTACG GACNNNGTC CCANNNNNTGG TCCNGGA GGCC ACCGGT CGATCG GAGTC(4/5) CACGTG GTTTAAAC CACGTG (7/12)GAAC(N)5CTC(13/8) GAGTC(4/5) YACGTR RGGWCCY ACATGT GACNNNNGTC ATTAAT TTATAA GAGCTC AACGTT FastDigest enzyme FastDigest MssI FastDigest MfeI (MunI) FastDigest Mva1269I
Page Conventional enzyme 48 45 49 48 25 57 124 49 38 49 50 50 60 44 49 50 22 51 51 51 52 52 39 52 53 27 14 53 64 69 29 16 31 64 59 49 49 53 67 26 57 54 54 40 13 57 46 54 48 54 MssI (PmeI) MunI (MfeI) Mva1269I (BsmI) [EcoRIIm](CCWGG), MvaI (BstNI) Bsh1236I (BstUI) PvuI HpyF10VI (MwoI)m PdiI (NaeI) [EheI (SfoI)](GGCGCC), [SspDI (KasI)](GGCGCC) BcnI (NciI) NcoI NdeI Bsp143I (Sau3AI)m, MboIm [PdiI (NaeI)](GCCGGC) NheI, [BspOI (BmtI)](GCTAGC) Hin1II (NlaIII) BspLI (NlaIV)m NmuCI (Tsp45I) NotI Bsp68I (NruI) NsbI (FspI) Mph1103I (NsiI) XceI (NspI) Bsp119I (BstBI) OliI (AleI) PacI PaeI (SphI) XhoI PagI (BspHI) SgsI (AscI) PasI PauI (BssHII) Eco147I (StuI) PscI (PciI) LguI (SapI) Mva1269I (BsmI) PdiI (NaeI) PdmI (XmnI) PfeI (TfiI) Pfl23II (BsiWI) PsyI (Tth111I) Van91I (PflMI) PfoI BsuRI (HaeIII) BshTI (AgeI) PvuI [SchI (MlyI)](GAGTC(5/5)) Eco72I (PmlI) MssI (PmeI)m Eco72I (PmlI) PpiI [SchI (MlyI)](GAGTC(5/5)) Ppu21I (BsaAI) Psp5II (PpuMI) PscI (PciI) BoxI (PshAI) VspI (AseI) AanI (PsiI) [Ecl136II (EcoICRI)](GAGCTC), SacI Psp1406I (AclI)
Page 130 130 131 116 130 97 141 124 136 117 150 88 131 132 99 127 136 132 100 120 100 133 133 98 133 129 156 99 134 134 134 156 135 147 135 135 115 138 125 131 136 136 136 137 140 154 137 103 101 141 144 113 130 113 138 144 138 139 138 91 154 80 109 142 139
FastDigest MvaI FastDigest Bsh1236I FastDigest PvuI FastDigest HpyF10VIm FastDigest NaeI (PdiI)
[FastDigest EheI](GGCGCC) FastDigest NciI (BcnI) FastDigest NcoI FastDigest NdeI FastDigest Sau3AI (Bsp143I)m, FastDigest MboIm [FastDigest NaeI (PdiI)](GCCGGC) FastDigest NheI, [FastDigest BmtI (BspOI)](GCTAGC) FastDigest NlaIII (Hin1II) FastDigest NlaIV (BspLI)m
Commercially available isoschizomers from other vendors PmeIm MfeI BsaMI, BsmI, PctI [AjnI], BseBI, Bst2UI, BstNI, BstOI, [Psp6I], [PspGIm] AccII, BspFNI, BstFNI, BstUI BpvUI, Ple19I BstMWI [MroNI], NaeI, [NgoMIV] [BbeI], [DinI], [EgeI], [KasI], Mly113I, [SfoIm] AsuC2I, BpuMI, NciI Bsp19I FauNDI BfuCIm, BssMI, [BstKTIm], BstMBI, DpnII, Kzo9Im, Sau3AIm MroNI, [NaeI] AsuNHI, [BmtI]m [CviAII], FaeI, [FatIm], Hsp92II, NlaIII BmiI, NlaIV, PspN4I Tsp45I CciNI BtuMI, NruI Acc16I, AviII, FspI EcoT22Im, NsiI, Zsp2I BstNSI, NspI AsuIIm, Bpu14I, BspT104I, BstBIm, Csp45Im, SfuIm AleI BbuI, SphI Sfr274I, SlaI, StrI, TliI CciI, BspHIm, RcaI AscI BsePI, BssHII AatI, SseBI, StuI BspQI, SapI BsaMI, BsmI [MroNI], NaeI, [NgoMIV] Asp700I, MroXI, XmnIm TfiI BsiWI, PspLI AspI, Tth111I AccB7I, BasI, PflMI BshFI, BsnI, BspANI, HaeIII AgeI, AsiGI, CspAI BpvUI, MvrI [MlyIm], PpsI AcvI, BbrPI, PmlI, PspCI AcvI, BbrPI, PmaCI, PmlI, PspCI [MlyI], PleI BsaAI, BstBAI PpuMI, PspPPI PciI BstPAI, PshAI AseI PsiI [Eco53kI], [EcoICRI], SstI AclI (continued on next page)
FastDigest NmuCI FastDigest NotI FastDigest NruI (RruI) FastDigest FspI (NsbI) FastDigest NsiI (Mph1103I) FastDigest NspI (XceI) FastDigest Bsp119I FastDigest AleI (OliI) FastDigest PacI FastDigest SphI (PaeI) FastDigest XhoI FastDigest BspHI (PagI) FastDigest AscI (SgsI) FastDigest BssHII (PteI) FastDigest StuI (Eco147I) FastDigest SapI (LguI) FastDigest Mva1269I FastDigest NaeI (PdiI) FastDigest PdmI FastDigest TfiI (PfeI) FastDigest BsiWI (Pfl23II) FastDigest PsyI FastDigest PflMI (Van91I) FastDigest PfoI FastDigest HaeIII (BsuRI) FastDigest AgeI (BshTI) FastDigest PvuI [FastDigest MlyI (SchI)](GAGTC(5/5)) FastDigest PmlI (Eco72I) FastDigest MssIm FastDigest PmlI (Eco72I)
[FastDigest MlyI (SchI)](GAGTC(5/5)) 46 23 FastDigest BsaAI (Ppu21I) 55 FastDigest PpuMI (Psp5II) FastDigest PshAI (BoxI) FastDigest AseI (VspI) FastDigest PsiI (AanI) [FastDigest Ecl136II](GAGCTC), FastDigest SacI FastDigest AclI (Psp1406I) 55 17 55 35 58 12
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Enzyme Psp5II Psp6I PspCI PspEI PspFI PspGI PspLI PspN4I PspOMI PspPI PspPPI PspXI PsrI PstI PsuI PsyI PvuI PvuII RcaI RgaI RigI RsaI RsaNI RseI Rsr2I RsrII SacI SacII SalI SanDI SapI SatI Sau3AI Sau96I SbfI ScaI SchI ScrFI SdaI SduI SetI SexAI SfaAI SfaNI SfcI SfiI SfoI Sfr274I Sfr303I SgeI SfuI SgfI SgrAI SgrBI SgrDI SgsI SinI SlaI
Specificity 53 RGGWCCY CCWGG CACGTG GGTNACC CCCAGC(-1/-5) CCWGG CGTACG GGNNCC GGGCCC GGNCC RGGWCCY VCTCGAGB (7/12)GAAC(N)6TAC(12/7) CTGCAG RGATCY GACNNNGTC CGATCG CAGCTG TCATGA GCGATCGC GGCCGGCC GTAC GTAC CAYNNNNRTG CGGWCCG CGGWCCG GAGCTC CCGCGG GTCGAC GGGWCCC GCTCTTC(1/4) GCNGC GATC GGNCC CCTGCAGG AGTACT GAGTC(5/5) CCNGG CCTGCAGG GDGCHC ASST ACCWGGT GCGATCGC GCATC(5/9) CTRYAG GGCCNNNNNGGCC GGCGCC CTCGAG CCGCGG m5CNNG(9/13) TTCGAA GCGATCGC CRCCGGYG CCGCGG CGTCGACG GGCGCGCC GGWCC CTCGAG
FastDigest enzyme FastDigest PpuMI (Psp5II) [FastDigest MvaI](CCWGG) FastDigest PmlI (Eco72I) FastDigest Eco91I FastDigest PspFI [FastDigest MvaIm](CCWGG) FastDigest BsiWI (Pfl23II) FastDigest NlaIV (BspLI) [FastDigest ApaI](GGGCCC), FastDigest Bsp120I FastDigest Sau96I (Cfr13I) FastDigest PpuMI (Psp5II)
Page Conventional enzyme 55 48 54 36 56 130 26 51 16 28 60 55 Psp5II (PpuMI) EcoRII, [MvaI (BstNI)](CCWGG) Eco72I (PmlI) Eco91I (BstEII) EcoRII, [MvaI (BstNI)m](CCWGG) Pfl23II (BsiWI) BspLI (NlaIV) [ApaI](GGGCCC), Bsp120I (PspOMI) Cfr13I (Sau96I) Psp5II (PpuMI)
Page 139 116 130 113 114 116 130 137 100 85 99 106 139
Commercially available isoschizomers from other vendors PpuMI, PspPPI AjnI, [BseBI], [Bst2UI], [BstNI], [BstOI], PspGI AcvI, BbrPI, PmaCI, PmlI BstEII, BstPI, EcoO65I GsaI, [BseYI] AjnIm, [BseBI], [Bst2UIm], [BstNIm], [BstOIm], Psp6I BsiWI BmiI, NlaIV
AspS9I, [BmgT120I], Sau96I PpuMI
FastDigest PstI FastDigest PsuI FastDigest PsyI FastDigest PvuI FastDigest PvuII FastDigest BspHI (PagI) FastDigest AsiSI (SfaAI) [FastDigest Csp6Im](GTAC), FastDigest RsaI FastDigest Csp6I, [FastDigest RsaI](GTAC) FastDigest MslI (RseI) FastDigest RsrII (CpoI) FastDigest RsrII (CpoI) [FastDigest Ecl136IIm](GAGCTC), FastDigest SacI FastDigest SalI FastDigest SanDI (KflI) FastDigest SapI (LguI) FastDigest Fnu4HI (SatI) FastDigest Sau3AI (Bsp143I), FastDigest MboIm FastDigest Sau96I (Cfr13I) FastDigest SbfI (SdaI) FastDigest ScaI FastDigest MlyI (SchI) FastDigest ScrFI (Bme1390I) FastDigest SbfI (SdaI) FastDigest Bsp1286I (SduI) FastDigest SexAI (CsiI)m FastDigest AsiSI (SfaAI) FastDigest SfaNI (BmsI) FastDigest SfcI (BfmI) FastDigest SfiI FastDigest EheIm FastDigest XhoI
56 56 57 57 57 29 17 32 58 32 58 47 58 58 35 58 59 59 59 38 60 44 60 60 61 46 61 60 28 61 17 62 62 62 38 69
PstI PsuI (BstYI) PsyI (Tth111I) PvuI PvuII PagI (BspHI) SfaAI (AsiSI) [Csp6I (CviQI)m](GTAC), RsaI Csp6I (CviQI), [RsaI](GTAC) RseI (MslI) CpoI (RsrII) CpoI (RsrII) [Ecl136II (EcoICRI)m](GAGCTC), SacI Cfr42I (SacII) SalI LguI (SapI) SatI (Fnu4HI) Bsp143I (Sau3AI), MboIm Cfr13I (Sau96I) SdaI (SbfI) ScaI SchI (MlyI) Bme1390I (ScrFI) SdaI (SbfI) SduI (Bsp1286I)
140 140 140 141 141 135 145 107 141 107 141 142 106 106 109 142 106 143 125 143 99 127 106 144 144 144 91 144 145
BspMAI BstX2I, BstYI, MflIm, XhoII AspI, PflFI, Tth111I BpvUI, MvrI, Ple19I BspHI, CciI AsiSI, SgfI FseI AfaI, [CviQIm], [RsaNI] [AfaI], CviQI MslI, SmiMI CspI, RsrII CspI, Rsr2I, RsrII [Eco53kIm], [EcoICRI], Psp124BI, SstI KspI, Sfr303I, SgrBI, SstII SanDI BspQI, PciSI, SapI Fnu4HI, Fsp4HI, ItaIm BfuCIm, BssMI, [BstKTIm], BstMBI, DpnIIm, Kzo9I, NdeIIm AspS9I, [BmgT120I], PspPI, Sau96I SbfI, Sse8387I AssI, BmcAI, ZrmI MlyI, [PleI], [PpsI], SchI BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I] SbfI, Sse8387I Bsp1286I, MhlI MabI m, SexAI AsiSI, RgaI, SgfI BstSFI, SfcI [BbeIm], DinI, EgeI, [KasIm], [Mly113I], [NarIm] PaeR7I, SlaI, StrI, TliI KspI, SacII, SgrBI, SstII AsuII, Bpu14I, BspT104Im, BstBIm, Csp45I, NspVm AsiSI, RgaI KspI, SacII, Sfr303I, SstII AscI, PalAI AvaII, Bme18I, VpaK11BI PaeR7I, Sfr274I, StrI, TliI (continued on next page)
SfaAI (AsiSI) LweI (SfaNI) BfmI (SfcI) SfiI EheI (SfoI)m, [SspDI (KasI)](GGCGCC) XhoI Cfr42I (SacII) SgeI Bsp119I (BstBI)m SfaAI (AsiSI) Cfr42I (SacII) SgrDI SgsI (AscI) Eco47I (AvaII) XhoI
145 126 89 146 117 150 156 106 146 99 145 106 147 147 111 156
FastDigest Bsp119Im FastDigest AsiSI (SfaAI)
27 17
FastDigest AscI (SgsI) FastDigest AvaII (Eco47I) FastDigest XhoI
16 18 69
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Enzyme SmaI SmiI SmiMI SmlI SmoI SmuI SnaBI SpeI SphI SrfI Sse8387I Sse9I SseBI SsiI SspDI SspI SstI SstII StrI StuI StyD4I StyI SwaI TaaI TaiI TaqI TaqII TasI TatI TauI TfiI TliI Tru1I Tru9I TscAI TseI TsoI Tsp45I Tsp509I TspDTI TspEI TspGWI TspMI TspRI TstI Tth111I Van91I Vha464I VneI VpaK11BI VspI XagI XapI XbaI XceI XcmI XhoI XhoII XmaCI Specificity 53 CCCGGG ATTTAAAT CAYNNNNRTG CTYRAG CTYRAG CCCGC(4/6) TACGTA ACTAGT GCATGC GCCCGGGC CCTGCAGG AATT AGGCCT CCGC(-3/-1) GGCGCC AATATT GAGCTC CCGCGG CTCGAG AGGCCT CCNGG CCWWGG ATTTAAAT ACNGT ACGT TCGA GACCGA(11/9), CACCCA(11/9) AATT WGTACW GCSGC GAWTC CTCGAG TTAA TTAA CASTG(2/-7) GCWGC TARCCA(11/9) GTSAC AATT ATGAA(11/9) AATT ACGGA(11/9) CCCGGG CASTGNN(2/-7) (8/13)CAC(N)6TCC(12/7) GACNNNGTC CCANNNNNTGG CTTAAG GTGCAC GGWCC ATTAAT CCTNNNNNAGG RAATTY TCTAGA RCATGY CCANNNNNNNNNTGG CTCGAG RGATCY CCCGGG FastDigest enzyme FastDigest SmaI FastDigest SwaI (SmiI) FastDigest MslI (RseI)
Page Conventional enzyme 63 65 47 [Cfr9I (XmaI)m](CCCGGG), SmaI SmiI (SwaI) RseI (MslI) SmoI (SmlI) SmoI (SmlI) SmuI (FauI) Eco105I (SnaBI) BcuI (SpeI) PaeI (SphI) SdaI (SbfI) TasI (Tsp509I) Eco147I (StuI) SsiI (AciI) [EheI (SfoI)](GGCGCC), SspDI (KasI) SspI [Ecl136II (EcoICRI)m](GAGCTC), SacI Cfr42I (SacII) XhoI Eco147I (StuI) [Bme1390I (ScrFI)](CCNGG) Eco130I (StyI) SmiI (SwaI) TaaI (HpyCH4III) TaiI (MaeII) TaqI
Page 105 147 148 142 148 148 148 114 88 134 144 151 115 149 117 150 149 109 142 106 156 115 91 114 148 150 150 151
Commercially available isoschizomers from other vendors [TspMI], [XmaCI], [XmaIm] SwaI MslI SmlI FauI BstSNI, SnaBI AhlI, SpeI BbuI, SphI SbfI Tsp509I, TspEI AatI, PceI, StuI AciI, BspACI [BbeI], [DinI], [EgeI], KasI, [Mly113I], [NarI], [SfoI] [Eco53kI], [EcoICRI], Psp124BI KspI, SacII, Sfr303I, SgrBI PaeR7I, Sfr274I, SlaI, TliI AatI, PceI, SseBI, StuI [BmrFI], BssKI, BstSCI, [MspR9I], [ScrFI] BssT1I, EcoT14I, ErhI, StyI SwaI Bst4CI, HpyCH4III [HpyCH4IVm], [MaeII]
FastDigest SnaBI (Eco105I) FastDigest SpeI (BcuI) FastDigest SphI (PaeI) FastDigest SbfI (SdaI) FastDigest Tsp509I (TasI) FastDigest StuI (Eco147I) FastDigest AciI (SsiI) [FastDigest EheI](GGCGCC) FastDigest SspI [FastDigest Ecl136IIm](GAGCTC), FastDigest SacI FastDigest XhoI FastDigest StuI (Eco147I) [FastDigest ScrFI (Bme1390I)](CCNGG) FastDigest StyI (Eco130I) FastDigest SwaI (SmiI) FastDigest TaaI FastDigest TaiI FastDigest TaqI
63 63 64 60 68 64 12 38 64 35 58 69 64 61 65 65 150 66 66
FastDigest Tsp509I (TasI) FastDigest TatI FastDigest TauI FastDigest TfiI (PfeI) FastDigest XhoI FastDigest MseI (SaqAI), FastDigest Tru1I FastDigest MseI (SaqAI), FastDigest Tru1I FastDigest TspRI (TscAI)
68 66 67 67 69 47 67 47 67 68
TasI (Tsp509I) TatI TauI PfeI (TfiI) XhoI Tru1I (MseI) Tru1I (MseI) TscAI (TspRI) TsoI NmuCI (Tsp45I) TasI (Tsp509I) TasI (Tsp509I) Cfr9I (XmaI)m, [SmaI](CCCGGG) TscAI (TspRI) TstI PsyI (Tth111I) Van91I (PflMI) BspTI (AflII) Alw44I (ApaLI) Eco47I (AvaII) VspI (AseI) XagI (EcoNI) XapI (ApoI) XbaI XceI (NspI) XhoI PsuI (BstYI) Cfr9I (XmaI), [SmaI](CCCGGG)
151 151 152 136 156 152 152 153 153 133 151 151 105 147 153 154 140 154 101 85 111 154 155 155 155 156 156 140 105 147
Sse9I, Tsp509I, TspEI
TfiI PaeR7I, Sfr274I, SlaI, StrI MseI, Tru9I MseI TspRI ApeKI
FastDigest NmuCI FastDigest Tsp509I (TasI) FastDigest Tsp509I (TasI) [FastDigest SmaI](CCCGGG) FastDigest TspRI (TscAI) FastDigest PsyI FastDigest PflMI (Van91I) FastDigest AflII (BspTI) FastDigest ApaLI (Alw44I) FastDigest AvaII (Eco47I) FastDigest AseI (VspI) FastDigest EcoNI (XagI) FastDigest XapI FastDigest XbaI FastDigest NspI (XceI) FastDigest XhoI FastDigest PsuI [FastDigest SmaI](CCCGGG)
51 68 68 63 68 57 54 13 16 18 17 36 68 69 53 69 56 63
Sse9I, Tsp509I, TspEI Sse9I, Tsp509I XmaCI, XmaIm TspRI AspI, PflFI AccB7I, BasI, PflMI AflII, BfrI, Bst98I, BstAFI, MspCI ApaLI AvaII, Bme18I, SinI AseI, PshBI BstENI, EcoNI AcsI, ApoI BstNSI, NspI PaeR7I, Sfr274I, SlaI, StrI, TliI BstX2I, BstYI, MflIm, TspMI, XmaI (continued on next page)
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Enzyme XmaI XmaJI XmiI XmnI XspI ZraI ZrmI Zsp2I
Specificity 53 CCCGGG CCTAGG GTMKAC GAANNNNTTC CTAG GACGTC AGTACT ATGCAT
FastDigest enzyme [FastDigest SmaIm](CCCGGG) FastDigest AvrII (XmaJI) FastDigest AccI (XmiI) FastDigest PdmIm FastDigest BfaI (FspBI) [FastDigest AatIIm](GACGTC) FastDigest ScaI FastDigest NsiI (Mph1103I)
Page Conventional enzyme 63 18 11 53 20 11 144 52 Cfr9I (XmaI)m, [SmaIm](CCCGGG) XmaJI (AvrII) XmiI (AccI) PdmI (XmnI)m FspBI (BfaI) [AatIIm](GACGTC) ScaI Mph1103I (NsiI)
Page 105 147 156 157 136 119 81 144 129
Commercially available isoschizomers from other vendors TspMIm, XmaCI AspA2I, AvrII, BlnI AccI, FblI Asp700I, MroXI BfaI, MaeI AssI, BmcAI EcoT22I, NsiI
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Alphabetic List of Recognition Sequences
Table 1.26. Recognition sequences of restriction enzymes. Specificity 53 AACGTT AAGCTT (8/13)AAG(N)5CTT(13/8) AATATT AATT ACATGT ACCGGT ACCTGC(4/8)* ACCWGGT ACGCGT ACGGA(11/9)* ACGGC(12/14)* ACGT ACGT ACNGT (10/15)AC(N)4GTAYC(12/7)* (9/12)AC(N)5CTCC(10/7)* ACRYGT ACTAGT ACTGG(1/-1)* ACTGGG(5/4)* AGATCT AGCGCT AGCT AGGCCT AGTACT ASST ATCGAT ATGAA(11/9)* ATGCAT ATTAAT ATTTAAAT C (11/13)CAA(N)5GTGG(12/10)* CAATTG CACCCA(11/9)* CACCTGC(4/8)* CACGAG(-5/-1)* CACGTC(-3/-3)* CACGTG CACNNNGTG CACNNNNGTG (8/13)CAC(N)6TCC(12/7)* (0/2)CACTGC* CAGCAG(25/27)* CAGCTG CAGNNNCTG CASTG(2/-7) CATATG (13/9)CATCC* (0/2)CATCC* (14/10)CATCGC* CATG CATG CATG (0/2)CATTGC* CAYNNNNRTG (6/11)CCAA(N)7TTC(12/7)* (10/12)CCAC(N)5TTG(13/11)* FastDigest enzyme FastDigest AclI (Psp1406I) FastDigest HindIII Conventional enzyme Psp1406I (AclI) HindIII FalI FastDigest SspI SspI FastDigest Tsp509I (TasI) TasI (Tsp509I) PscI (PciI) FastDigest AgeI (BshTI) BshTI (AgeI) FastDigest BspMI (BveI) BveI (BspMI) FastDigest SexAI (CsiI) SexAI FastDigest MluI MluI TspGWI BceAI FastDigest TaiI TaiI (MaeII) MaeII FastDigest TaaI TaaI (HpyCH4III) BaeI BsaXI AflIII FastDigest SpeI (BcuI) BcuI (SpeI) FastDigest BseNI BseNI (BsrI) BfiI (BmrI) FastDigest BglII BglII FastDigest AfeI (Eco47III) Eco47III (AfeI) FastDigest AluI AluI FastDigest StuI (Eco147I) Eco147I (StuI) FastDigest ScaI ScaI SetI FastDigest ClaI (Bsu15I) Bsu15I (ClaI) TspDTI FastDigest NsiI (Mph1103I) Mph1103I (NsiI) FastDigest AseI (VspI) VspI (AseI) FastDigest SwaI (SmiI) SmiI (SwaI) CspCI MunI (MfeI) TaqII AarI BauI (BssSI) AjiI (BmgBI) Eco72I (PmlI) AdeI (DraIII) OliI (AleI) TstI BtsI EcoP15I PvuII CaiI (AlwNI) TscAI (TspRI) NdeI FokI BseGI (BtsCI) BtgZI Hin1II (NlaIII) FatI CviAII BseMI (BsrDI) RseI (MslI) AjuI CspCI
Specificity 53 (-1/1)CCAGT* CCANNNNNNNNNTGG CCANNNNNNTGG CCANNNNNTGG CCATC(4/5)* CCATGG CCCAGC(-5/-1)* CCCAGC(-1/-5)* (4/5)CCCAGT* CCCGC(4/6)* CCCGGG CCCGGG CCCWGGG CCGC(-3/-1)* CCGCGG CCGCTC(-3/-3)* CCGG CCNGG CCNGG CCNNGG CCNNNNNNNGG CCRYGG CCSGG CCTAGG CCTC(7/6)* CCTCAGC(-5/-2)* CCTCGAGG CCTGCAGG CCTNAGC(-5/-2)* CCTNAGG CCTNNNNNAGG CCTTC(6/5)* CCWGG CCWGG CCWWGG (10/12)CGA(N)6TGC(12/10)* CGATCG CGCCGGCG CGCG CGCGCGCG CGGCCG CGGWCCG CGRYCG CGTACG CGTCGACG CGTCTC(1/5)* CGWCG CMGCKG m5CNNG(9/13) (6/11)CRAA(N)6GTC(13/18)* CRCCGGYG CTAG (14/16)CTCAAG* CTCAG(9/7)* CTCAG(10/8)* (14/16)CTCCAG* (8/10)CTCCTC* CTCGAG
FastDigest enzyme FastDigest BseNI FastDigest BstXI FastDigest PflMI (Van91I) FastDigest NcoI FastDigest PspFI
FastDigest SmaI FastDigest AciI (SsiI) FastDigest BsrBI (MbiI) FastDigest HpaII FastDigest MspI FastDigest ScrFI (Bme1390I) FastDigest BsaJI (BseDI) FastDigest BslI (BseLI) FastDigest NciI (BcnI) FastDigest AvrII (XmaJI) FastDigest MnlI
FastDigest SbfI (SdaI) FastDigest Bpu10I FastDigest Bsu36I (Eco81I) FastDigest EcoNI (XagI)
FastDigest MfeI (MunI)
FastDigest MvaI FastDigest StyI (Eco130I) FastDigest PvuI FastDigest MreI FastDigest Bsh1236I FastDigest MauBI FastDigest EagI (Eco52I) FastDigest RsrII (CpoI) FastDigest BsiEI (Bsh1285I) FastDigest BsiWI (Pfl23II) FastDigest BsmBI (Esp3I)
FastDigest PmlI (Eco72I) FastDigest DraIII (AdeI) FastDigest AleI (OliI)
FastDigest PvuII FastDigest AlwNI (CaiI) FastDigest TspRI (TscAI) FastDigest NdeI FastDigest FokI FastDigest BseGI FastDigest NlaIII (Hin1II)
FastDigest BfaI (FspBI)
FastDigest BsrDI (BseMI) FastDigest MslI (RseI) FastDigest AjuI
FastDigest BspCNI (BseMII) FastDigest BpmI (GsuI) FastDigest XhoI
Conventional enzyme BseNI (BsrI) XcmI BstXI Van91I (PflMI) BccI NcoI BseYI GsaI BfiI (BmrI) SmuI (FauI) Cfr9I (XmaI) SmaI PasI SsiI (AciI) Cfr42I (SacII) MbiI (BsrBI) HpaII MspI (HpaII) StyD4I Bme1390I (ScrFI) BseDI (BsaJI) BseLI (BslI) BtgI BcnI (NciI) XmaJI (AvrII) MnlI BbvCI AbsI SdaI (SbfI) Bpu10I Eco81I (Bsu36I) XagI (EcoNI) HpyAV EcoRII MvaI (BstNI) Eco130I (StyI) BcgI PvuI MreI (Sse232I) Bsh1236I (BstUI) MauBI Eco52I (EagI) CpoI (RsrII) Bsh1285I (BsiEI) Pfl23II (BsiWI) SgrDI Esp3I (BsmBI) Hpy99I MspA1I SgeI ArsI SgrAI FspBI (BfaI) BpuEI BspCNI BseMII (BspCNI) GsuI (BpmI) BseRI XhoI
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Table 1.26.Recognition sequences of restriction enzymes.
1
Specificity 53 (19/21)CTCGGC* CTCGTG(-5/-1)* CTCTTC(1/4)* CTGAAG(16/14)* (7/9)CTGAG* (8/10)CTGAG* (14/16)CTGCAC* CTGCAG (27/25)CTGCTG* CTGGAG(16/14)* CTGRAG(16/14)* CTNAG CTRYAG CTTAAG (14/16)CTTCAG* CTTGAG(16/14)* (14/16)CTYCAG* CTYRAG CYCGRG CYCGRG G (7/12)GAAC(N)5CTC(13/8)* (7/12)GAAC(N)6TAC(12/7)*
Conventional enzyme NmeAIII BauI (BssSI) FastDigest EarI (Eam1104I) Eam1104I (EarI) FastDigest AcuI (Eco57I) Eco57I (AcuI) BspCNI FastDigest BspCNI (BseMII) BseMII (BspCNI) BsgI FastDigest PstI PstI EcoP15I FastDigest BpmI (GsuI) GsuI (BpmI) Eco57MI FastDigest DdeI (HpyF3I) HpyF3I (DdeI) FastDigest SfcI (BfmI) BfmI (SfcI) FastDigest AflII (BspTI) BspTI (AflII) FastDigest AcuI (Eco57I) Eco57I (AcuI) BpuEI Eco57MI SmoI (SmlI) FastDigest AvaI (Eco88I) Eco88I (AvaI) BmeT110I
FastDigest enzyme
Specificity 53 GATC Gm6ATC GATC (5/4)GATCC* (9/5)GATGC* (5/4)GATGG* GATNNNNATC GAWTC

(8/13-14)GAY(N)5VTC(13-14/8)*
FastDigest enzyme FastDigest Sau3AI (Bsp143I) FastDigest MboI FastDigest DpnI
FastDigest SfaNI (BmsI) FastDigest BsaBI (BseJI) FastDigest TfiI (PfeI) FastDigest BsrDI (BseMI) FastDigest BbvI (Lsp1109I) FastDigest BseXI FastDigest BspMI (BveI)
GCAATG(2/0)* GCAGC(8/12)* (8/4)GCAGGT* (8/4)GCAGGTG* GCAGTG(2/0)* (10/12)GCA(N)6TCG(12/10)* (10/12)GCA(N)6TGC(12/10) GCANNNNNTGC GCATC(5/9)* GCATGC (-1/1)GCATTC* GCCCGGGC GCCGAG(21/19)* GCCGGC GCCGGC (14/12)GCCGT* GCCNNNNNGGC GCGATCGC GCGATG(10/14)* GCGC GCGC GCCG GCGCGC GCGG(-3/-1)* GCGGCCGC (6/4)GCGGG* (10/5)GCGTC* GCNGC GCNGC GCNGC GCNNGC GCNNNNNNNGC GCSGC GCTAGC GCTAGC GCTCTTC(1/4)* GCTGAGG(-5/-2)* (12/8)GCTGC* GCTGGG(-5/-1)* GCTGGG(-1/-5)* GCTNAGC GCTNAGG(-5/-2)* GCWGC GDGCHC (7/10)GGAG(N)5GT(12/9)* (7/12)GGA(N)6GTG(13/8)*
(7/12-13)GGA(N)6GTTC(12-13/7)*
PpiI PsrI (7/12-13)GAAC(N)6TCC(12-13/7)* AloI FastDigest MboII MboII GAAGA(8/7)* GAAGAC(2/6)* FastDigest BbsI (BpiI) BpiI (BbsI) (4/1)GAAGAG* FastDigest EarI (Eam1104I) Eam1104I (EarI) (4/1)GAAGAGC* FastDigest SapI (LguI) LguI (SapI) (5/6)GAAGG* HpyAV (7/12)GAAG(N)6TAC(12/7)* BarI AjuI (7/12)GAA(N)7TTGG(11/6)* FastDigest AjuI FastDigest PdmI GAANNNNTTC PdmI (XmnI) FastDigest Mva1269I GAATGC(1/-1)* Mva1269I (BsmI) FastDigest EcoRI EcoRI GAATTC (8/13-14)GAB(N)5RTC(13-14/8)* Hin4I GACCGA(11/9)* TaqII GACGC(5/10)* FastDigest HgaI (CseI) CseI (HgaI) GACGTC ZraI FastDigest AatII AatII GACGTC GACGTG(-3/-3)* AjiI (BmgBI) FastDigest PsyI GACNNNGTC PsyI (Tth111I) GACNNNNGTC FastDigest PshAI (BoxI) BoxI (PshAI) FastDigest Eam1105I GACNNNNNGTC Eam1105I (AhdI) GACNNNNNNGTC FastDigest DrdI (AasI) AasI (DrdI) (8/13)GAC(N)6TTYG(11/6)* ArsI (5/5)GACTC* FastDigest MlyI (SchI) SchI (MlyI) (5/4)GACTC* PleI FastDigest Alw26I (5/1)GAGAC* Alw26I (BsmAI) FastDigest Eco31I (5/1)GAGACC* Eco31I (BsaI) (5/1)GAGACG* FastDigest BsmBI (Esp3I) Esp3I (BsmBI) GAGCGG(-3/-3)* FastDigest BsrBI (MbiI) MbiI (BsrBI) FastDigest SacI SacI GAGCTC FastDigest Ecl136II GAGCTC Ecl136II (EcoICRI) FastDigest MnlI MnlI (6/7)GAGG* GAGGAG(10/8)* BseRI FastDigest BplI BplI (8/13)GAG(N)5CTC(13/8) PpiI (8/13)GAG(N)5GTTC(12/7)* GAGTC(5/5)* FastDigest MlyI (SchI) SchI (MlyI) GAGTC(4/5)* PleI FastDigest HinfI HinfI GANTC GATATC FastDigest EcoRV (Eco32I) Eco32I (EcoRV)
FastDigest SfaNI (BmsI) FastDigest SphI (PaeI) FastDigest Mva1269I
FastDigest NaeI (PdiI) FastDigest BglI FastDigest AsiSI (SfaAI) FastDigest HinP1I (Hin6I) FastDigest HhaI FastDigest BssHII (PteI) FastDigest AciI (SsiI) FastDigest NotI FastDigest HgaI (CseI) FastDigest Fnu4HI (SatI)
FastDigest HpyF10VI FastDigest TauI FastDigest BmtI (BspOI) FastDigest NheI FastDigest SapI (LguI) FastDigest BbvI (Lsp1109I) FastDigest BseXI FastDigest PspFI FastDigest BlpI (Bpu1102I) FastDigest Bpu10I FastDigest Bsp1286I (SduI)
(5/6)GGATAC* GGATC(4/5)*
Conventional enzyme Bsp143I (Sau3AI) MboI DpnI BstKTI BspPI (AlwI) LweI (SfaNI) BccI BseJI (BsaBI) PfeI (TfiI) Hin4I BseMI (BsrDI) Lsp1109I (BbvI) BseXI (BbvI) BveI (BspMI) AarI BtsI BcgI AlfI BstAPI LweI (SfaNI) PaeI (SphI) Mva1269I (BsmI) SrfI NmeAIII NgoMIV PdiI (NaeI) BceAI BglI SfaAI (AsiSI) BtgZI Hin6I (HinP1I) HhaI GlaI PauI (BssHII) SsiI (AciI) NotI SmuI (FauI) CseI (HgaI) SatI (Fnu4HI) BisI BlsI Cac8I HpyF10VI (MwoI) TauI BspOI (BmtI) NheI LguI (SapI) BbvCI Lsp1109I (BbvI) BseXI (BbvI) BseYI GsaI Bpu1102I (BlpI) Bpu10I TseI SduI (Bsp1286I) BsaXI TstI AloI BfuI (BciVI) BspPI (AlwI)
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Table 1.26.Recognition sequences of restriction enzymes. Specificity 53 GGATCC GGATG(9/13)* GGATG(2/0)* GGCC GGCCGGCC GGCCNNNNNGGCC GGCGCC GGCGCC GGCGCC GGCGCC GGCGCGCC GGCGGA(11/9)* GGGAC(10/14)* GGGCCC GGGCCC GGGWCCC GGNCC GGNCC GGNNCC GGTACC GGTACC GGTCTC(1/5)* GGTGA(8/7)* GGTNACC GGWCC GGYRCC GKGCMC GRCGYC GRGCYC (7/12)GRTAC(N)4GT(15/10)* GTAC GTAC (7/12)GTA(N)6CTTC(12/7)* (7/12)GTA(N)6GTTC(12/7)* GTATAC GTATCC(6/5)* (14/10)GTCCC* GTCGAC GTCTC(1/5)* (6/2)GTCTTC* GTGCAC GTGCAG(16/14)* GTMKAC GTNAC GTNNAC GTSAC GTTAAC GTTTAAAC (18/20)GTYGGA* GTYRAC GWGCWC R RAATTY RCATGY RCCGGY RGATCY RGCGCY RGCY RGGNCCY RGGWCCY RTGCGCAY FastDigest enzyme FastDigest BamHI FastDigest FokI FastDigest BseGI FastDigest HaeIII (BsuRI) FastDigest SfiI FastDigest EheI Conventional enzyme BamHI FokI BseGI (BtsCI) BsuRI (HaeIII) FseI SfiI EheI (SfoI) NarI SspDI (KasI) BbeI SgsI (AscI) EciI FaqI (BsmFI) Bsp120I (PspOMI) ApaI SanDI Cfr13I (Sau96I) BmgT120I BspLI (NlaIV) KpnI Acc65I (Asp718I) Eco31I (BsaI) HphI Eco91I (BstEII) Eco47I (AvaII) BshNI (BanI) BseSI (Bme1580I) Hin1I (BsaHI) Eco24I (BanII) BaeI RsaI Csp6I (CviQI) BarI PsrI Bst1107I (BstZ17I) BfuI (BciVI) FaqI (BsmFI) SalI Alw26I (BsmAI) BpiI (BbsI) Alw44I (ApaLI) BsgI XmiI (AccI) MaeIII Hpy8I (MjaIV) NmuCI (Tsp45I) KspAI (HpaI) MssI (PmeI) MmeI HincII (HindII) Alw21I (BsiHKAI)
FastDigest AscI (SgsI) FastDigest BsmFI (FaqI) FastDigest Bsp120I FastDigest ApaI FastDigest SanDI (KflI) FastDigest Sau96I (Cfr13I) FastDigest NlaIV (BspLI) FastDigest KpnI FastDigest Acc65I FastDigest Eco31I FastDigest Eco91I FastDigest AvaII (Eco47I) FastDigest BanI (BshNI) FastDigest Bme1580I (BseSI) FastDigest BsaHI (Hin1I)
Specificity 53 T TACGTA TARCCA(11/9)* (7/8)TCACC* TCATGA (9/11)TCCGCC* TCCGGA (9/11)TCCGT* (9/11)TCGGTC* TCCNGGA TCCRAC(20/18)* TCGA TCGCGA TCNGA TCNNGA TCTAGA (7/8)TCTTC* (10/12)TGA(N)6TCA(12/10) TGATCA TGCA TGCGCA TGGCCA (9/11)TGGGTG* (9/11)TGGYTA* TGTACA TTAA TTAATTAA TTATAA (9/11)TTCAT* TTCGAA TTSAA TTTAAA V VCTCGAGB W WCCGGW WGTACW Y YACGTR YATR YGGCCR
FastDigest enzyme
Conventional enzyme
FastDigest RsaI FastDigest Csp6I
FastDigest SnaBI (Eco105I) Eco105I (SnaBI) TsoI HphI FastDigest BspHI (PagI) PagI (BspHI) EciI FastDigest Kpn2I Kpn2I (BspEI) TspGWI TaqII FastDigest PfoI PfoI MmeI FastDigest TaqI TaqI FastDigest NruI (RruI) Bsp68I (NruI) Hpy188I Hpy188III FastDigest XbaI XbaI FastDigest MboII MboII BdaI FastDigest BclI BclI HpyCH4V FastDigest FspI (NsbI) NsbI (FspI) FastDigest MscI (MlsI) MlsI (MscI) TaqII TsoI FastDigest MscI (MlsI) FastDigest Bsp1407I Bsp1407I (BsrGI) FastDigest MseI (SaqAI) Tru1I (MseI) FastDigest Tru1I FastDigest PacI PacI FastDigest PsiI (AanI) AanI (PsiI) TspDTI FastDigest Bsp119I Bsp119I (BstBI) AgsI FastDigest DraI DraI PspXI BsaWI TatI
FastDigest BstZ17I (Bst1107I) FastDigest BsmFI (FaqI) FastDigest SalI FastDigest Alw26I FastDigest BbsI (BpiI) FastDigest ApaLI (Alw44I) FastDigest AccI (XmiI) FastDigest Hpy8I FastDigest NmuCI FastDigest HpaI (KspAI) FastDigest MssI
FastDigest TatI
FastDigest BsaAI (Ppu21I) Ppu21I (BsaAI) FaiI CfrI (EaeI)
Note * Asymetric sequences.
Single letter code

FastDigest HincII FastDigest Alw21I FastDigest XapI FastDigest NspI (XceI) FastDigest BsrFI (Cfr10I) FastDigest PsuI FastDigest HaeII (BfoI)
XapI (ApoI) XceI (NspI) Cfr10I (BsrFI) PsuI (BstYI) HaeII CviJI FastDigest EcoO109I EcoO109I (DraII) FastDigest PpuMI (Psp5II) Psp5II (PpuMI) FastDigest FspAI FspAI
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Enzyme Recognizing 2 bp Long Targets

Table 1.27. Enzyme recognizing 2 bp long targets.
Specificity 53 m5CNNG(9/13) FastDigest enzyme Conventional enzyme SgeI
Alphabetic List of Enzymes Recognizing 5 bp Long Targets

Table 1.29. Enzymes recognizing 5 bp long targets.
Specificity 53 ACGGA(11/9)* ACGGC(12/14)* ACTGG(1/-1)* ATGAA(11/9)* CASTG(2/-7) (13/9)CATCC* (0/2)CATCC* (-1/1)CCAGT* CCATC(4/5)* CCCGC(4/6)* CCSGG CCTTC(6/5)* CCWGG CCWGG CGWCG CTCAG(9/7)* CTCAG(10/8)* (7/9)CTGAG* (8/10)CTGAG* GAAGA(8/7)* (5/6)GAAGG* GACGC(5/10)* (5/5)GACTC* (5/4)GACTC* (5/1)GAGAC* GAGTC(5/5)* GAGTC(4/5)* (5/4)GATCC* (9/5)GATGC* (5/4)GATGG* GAWTC GCAGC(8/12)* GCATC(5/9)* (14/12)GCCGT* (6/4)GCGGG* (10/5)GCGTC* GCSGC (12/8)GCTGC* GCWGC GGATC(4/5)* GGATG(9/13)* GGATG(2/0)* GGGAC(10/14)* GGTGA(8/7)* GGWCC (14/10)GTCCC* GTCTC(1/5)* GTSAC (7/8)TCACC* (9/11)TCCGT* (7/8)TCTTC* (9/11)TTCAT* TTSAA FastDigest enzyme Conventional enzyme TspGWI BceAI BseNI (BsrI) TspDTI TscAI (TspRI) FokI BseGI (BtsCI) BseNI (BsrI) BccI SmuI (FauI) BcnI (NciI) HpyAV EcoRII MvaI (BstNI) Hpy99I BspCNI BseMII (BspCNI) BspCNI BseMII (BspCNI) MboII HpyAV CseI (HgaI) SchI (MlyI) PleI Alw26I (BsmAI) SchI (MlyI) PleI BspPI (AlwI) LweI (SfaNI) BccI PfeI (TfiI) Lsp1109I (BbvI) BseXI (BbvI) LweI (SfaNI) BceAI SmuI (FauI) CseI (HgaI) TauI Lsp1109I (BbvI) BseXI (BbvI) TseI BspPI (AlwI) FokI BseGI (BtsCI) FaqI (BsmFI) HphI Eco47I (AvaII) FaqI (BsmFI) Alw26I (BsmAI) NmuCI (Tsp45I) HphI TspGWI MboII TspDTI AgsI

Specificity 53 AATT ACGT ACGT ACNGT AGCT ASST CATG CATG CATG CCGC(-3/-1)* CCGG CCNGG CCNGG CCNNGG CCNNNNNNNGG CCTC(7/6)* CGCG CTAG CTNAG (6/7)GAGG* GANTC GATC Gm6ATC GATC GCGC GCGC GCGC GCGG(-3/-1)* GCNGC GCNGC GCNGC GCNNGC GCNNNNNNNGC GGCC GGNCC GGNCC GGNNCC GTAC GTAC GTNAC GTNNAC RGCY TCGA TCNGA TCNNGA TGCA TTAA YATR FastDigest enzyme FastDigest Tsp509I (TasI) FastDigest TaiI FastDigest TaaI FastDigest AluI FastDigest NlaIII (Hin1II) Conventional enzyme TasI (Tsp509I) TaiI (MaeII) MaeII TaaI (HpyCH4III) AluI SetI Hin1II (NlaIII) FatI CviAII SsiI (AciI) HpaII MspI (HpaII) StyD4I Bme1390I (ScrFI) BseDI (BsaJI) BseLI (BslI) MnlI Bsh1236I (BstUI) FspBI (BfaI) HpyF3I (DdeI) MnlI HinfI MboI Bsp143I (Sau3AI) DpnI BstKTI Hin6I (HinP1I) HhaI GlaI SsiI (AciI) SatI (Fnu4HI) BisI BlsI Cac8I HpyF10VI (MwoI) BsuRI (HaeIII) Cfr13I (Sau96I) BmgT120I BspLI (NlaIV) RsaI Csp6I (CviQI) MaeIII Hpy8I (MjaIV) CviJI TaqI Hpy188I Hpy188III HpyCH4V Tru1I (MseI) FaiI
FastDigest BseNI FastDigest TspRI (TscAI) FastDigest FokI FastDigest BseGI FastDigest BseNI
FastDigest NciI (BcnI)
FastDigest MvaI
FastDigest AciI (SsiI) FastDigest HpaII FastDigest MspI FastDigest ScrFI (Bme1390I) FastDigest BsaJI (BseDI) FastDigest BslI (BseLI) FastDigest MnlI FastDigest Bsh1236I FastDigest BfaI (FspBI) FastDigest DdeI (HpyF3I) FastDigest MnlI FastDigest HinfI FastDigest MboI FastDigest Sau3AI (Bsp143I) FastDigest DpnI FastDigest HinP1I (Hin6I) FastDigest HhaI FastDigest AciI (SsiI) FastDigest Fnu4HI (SatI)
FastDigest BspCNI (BseMII) FastDigest BspCNI (BseMII) FastDigest MboII FastDigest HgaI (CseI) FastDigest MlyI (SchI) FastDigest Alw26I FastDigest MlyI (SchI)
FastDigest SfaNI (BmsI) FastDigest TfiI (PfeI) FastDigest BbvI (Lsp1109I) FastDigest BseXI FastDigest SfaNI (BmsI)
FastDigest HgaI (CseI) FastDigest TauI FastDigest BbvI (Lsp1109I) FastDigest BseXI
FastDigest HpyF10VI FastDigest HaeIII (BsuRI) FastDigest Sau96I (Cfr13I) FastDigest NlaIV (BspLI) FastDigest RsaI FastDigest Csp6I FastDigest Hpy8I FastDigest TaqI
FastDigest FokI FastDigest BseGI FastDigest BsmFI (FaqI) FastDigest AvaII (Eco47I) FastDigest BsmFI (FaqI) FastDigest Alw26I FastDigest NmuCI
FastDigest MboII
FastDigest MseI (SaqAI) FastDigest Tru1I
Single letter code

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Specificity 53 AACGTT AAGCTT (8/13)AAG(N)5CTT(13/8) AATATT ACATGT ACCGGT ACCTGC(4/8)* ACGCGT (9/12)AC(N)5CTCC(10/7)* ACRYGT ACTAGT ACTGGG(5/4)* AGATCT AGCGCT AGGCCT AGTACT ATCGAT ATGCAT ATTAAT CAATTG CACCCA(11/9)* CACGAG(-5/-1)* CACGTC(-3/-3)* CACGTG CACNNNGTG CACNNNNGTG (8/13)CAC(N)6TCC(12/7)* (0/2)CACTGC* CAGCAG(25/27)* CAGCTG CAGNNNCTG CATATG (14/10)CATCGC* (0/2)CATTGC* CAYNNNNRTG CCANNNNNNNNNTGG CCANNNNNNTGG CCANNNNNTGG CCATGG CCCAGC(-5/-1)* CCCAGC(-1/-5)* (4/5)CCCAGT* CCCGGG CCCGGG CCGCGG CCGCTC(-3/-3)* CCRYGG CCTAGG CCTNAGC(-5/-2)* CCTNAGG CCTNNNNNAGG CCWWGG (10/12)CGA(N)6TGC(12/10)* CGATCG CGGCCG CGRYCG CGTACG CGTCTC(1/5)* CMGCKG (14/16)CTCAAG* (14/16)CTCCAG* (8/10)CTCCTC* CTCGAG (19/21)CTCGGC* FastDigest enzyme FastDigest AclI (Psp1406I) FastDigest HindIII FastDigest SspI FastDigest AgeI (BshTI) FastDigest BspMI (BveI) FastDigest MluI Conventional enzyme Psp1406I (AclI) HindIII FalI SspI PscI (PciI) BshTI (AgeI) BveI (BspMI) MluI BsaXI AflIII BcuI (SpeI) BfiI (BmrI) BglII Eco47III (AfeI) Eco147I (StuI) ScaI Bsu15I (ClaI) Mph1103I (NsiI) VspI (AseI) MunI (MfeI) TaqII BauI (BssSI) AjiI (BmgBI) Eco72I (PmlI) AdeI (DraIII) OliI (AleI) TstI BtsI EcoP15I PvuII CaiI (AlwNI) NdeI BtgZI BseMI (BsrDI) RseI (MslI) XcmI BstXI Van91I (PflMI) NcoI BseYI GsaI BfiI (BmrI) Cfr9I (XmaI) SmaI Cfr42I (SacII) MbiI (BsrBI) BtgI XmaJI (AvrII) Bpu10I Eco81I (Bsu36I) XagI (EcoNI) Eco130I (StyI) BcgI PvuI Eco52I (EagI) Bsh1285I (BsiEI) Pfl23II (BsiWI) Esp3I (BsmBI) MspA1I BpuEI GsuI (BpmI) BseRI XhoI NmeAIII Specificity 53 CTCGTG(-5/-1)* CTCTTC(1/4)* CTGAAG(16/14)* (14/16)CTGCAC* CTGCAG (27/25)CTGCTG* CTGGAG(16/14)* CTGRAG(16/14)* CTRYAG CTTAAG (14/16)CTTCAG* CTTGAG(16/14)* (14/16)CTYCAG* CTYRAG CYCGRG CYCGRG GAAGAC(2/6)* (4/1)GAAGAG* GAANNNNTTC GAATGC(1/-1)* GAATTC (8/13-14)GAB(N)5RTC(13-14/8)* GACCGA(11/9)* GACGTC GACGTC GACGTG(-3/-3)* GACNNNGTC GACNNNNGTC GACNNNNNGTC GACNNNNNNGTC (5/1)GAGACC* (5/1)GAGACG* GAGCGG(-3/-3)* GAGCTC GAGCTC GAGGAG(10/8)* (8/13)GAG(N)5CTC(13/8) GATATC GATNNNNATC (8/13-14)GAY(N)5VTC(13-14/8)* GCAATG(2/0)* (8/4)GCAGGT* GCAGTG(2/0)* (10/12)GCA(N)6TCG(12/10)* (10/12)GCA(N)6TGC(12/10) GCANNNNNTGC GCATGC (-1/1)GCATTC* GCCGAG(21/19)* GCCGGC GCCGGC GCCNNNNNGGC GCGATG(10/14)* GCGCGC GCTAGC GCTAGC GCTGGG(-5/-1)* GCTGGG(-1/-5)* GCTNAGC GCTNAGG(-5/-2)* GDGCHC (7/10)GGAG(N)5GT(12/9)* (7/12)GGA(N)6GTG(13/8)* (5/6)GGATAC* FastDigest enzyme FastDigest EarI (Eam1104I) FastDigest AcuI (Eco57I) FastDigest PstI FastDigest BpmI (GsuI) FastDigest SfcI (BfmI) FastDigest AflII (BspTI) FastDigest AcuI (Eco57I) Conventional enzyme BauI (BssSI) Eam1104I (EarI) Eco57I (AcuI) BsgI PstI EcoP15I GsuI (BpmI) Eco57MI BfmI (SfcI) BspTI (AflII) Eco57I (AcuI) BpuEI Eco57MI SmoI (SmlI) Eco88I (AvaI) BmeT110I BpiI (BbsI) Eam1104I (EarI) PdmI (XmnI) Mva1269I (BsmI) EcoRI Hin4I TaqII ZraI AatII AjiI (BmgBI) PsyI (Tth111I) BoxI (PshAI) Eam1105I (AhdI) AasI (DrdI) Eco31I (BsaI) Esp3I (BsmBI) MbiI (BsrBI) SacI Ecl136II (EcoICRI) BseRI BplI Eco32I (EcoRV) BseJI (BsaBI) Hin4I BseMI (BsrDI) BveI (BspMI) BtsI BcgI AlfI BstAPI PaeI (SphI) Mva1269I (BsmI) NmeAIII NgoMIV PdiI (NaeI) BglI BtgZI PauI (BssHII) BspOI (BmtI) NheI BseYI GsaI Bpu1102I (BlpI) Bpu10I SduI (Bsp1286I) BsaXI TstI BfuI (BciVI) (continued on next page)
FastDigest SpeI (BcuI) FastDigest BglII FastDigest AfeI (Eco47III) FastDigest StuI (Eco147I) FastDigest ScaI FastDigest ClaI (Bsu15I) FastDigest NsiI (Mph1103I) FastDigest AseI (VspI) FastDigest MfeI (MunI)
FastDigest AvaI (Eco88I) FastDigest BbsI (BpiI) FastDigest EarI (Eam1104I) FastDigest PdmI FastDigest Mva1269I FastDigest EcoRI
FastDigest PmlI (Eco72I) FastDigest DraIII (AdeI) FastDigest AleI (OliI)
FastDigest AatII FastDigest PsyI FastDigest PshAI (BoxI) FastDigest Eam1105I FastDigest DrdI (AasI) FastDigest Eco31I FastDigest BsmBI (Esp3I) FastDigest BsrBI (MbiI) FastDigest SacI FastDigest Ecl136II FastDigest BplI FastDigest EcoRV (Eco32I) FastDigest BsaBI (BseJI) FastDigest BsrDI (BseMI) FastDigest BspMI (BveI)
FastDigest PvuII FastDigest AlwNI (CaiI) FastDigest NdeI FastDigest BsrDI (BseMI) FastDigest MslI (RseI) FastDigest BstXI FastDigest PflMI (Van91I) FastDigest NcoI FastDigest PspFI
FastDigest SmaI FastDigest BsrBI (MbiI) FastDigest AvrII (XmaJI) FastDigest Bpu10I FastDigest Bsu36I (Eco81I) FastDigest EcoNI (XagI) FastDigest StyI (Eco130I) FastDigest PvuI FastDigest EagI (Eco52I) FastDigest BsiEI (Bsh1285I) FastDigest BsiWI (Pfl23II) FastDigest BsmBI (Esp3I)
FastDigest SphI (PaeI) FastDigest Mva1269I
FastDigest NaeI (PdiI) FastDigest BglI FastDigest BssHII (PfeI) FastDigest BmtI (BspOI) FastDigest NheI FastDigest PspFI FastDigest BlpI (Bpu1102I) FastDigest Bpu10I FastDigest Bsp1286I (SduI)
FastDigest BpmI (GsuI) FastDigest XhoI
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Table 1.30.Enzymes recognizing 6 bp long targets.
Specificity 53 GGATCC GGCGCC GGCGCC GGCGCC GGCGCC GGCGGA(11/9)* GGGCCC GGGCCC GGTACC GGTACC GGTCTC(1/5)* GGTNACC GGYRCC GKGCMC GRCGYC GRGCYC GTATAC GTATCC(6/5)* GTCGAC (6/2)GTCTTC* GTGCAC GTGCAG(16/14)* GTMKAC GTTAAC (18/20)GTYGGA* GTYRAC GWGCWC RAATTY RCATGY RCCGGY RGATCY RGCGCY RGGNCCY TACGTA TARCCA(11/9)* TCATGA (9/11)TCCGCC* TCCGGA TCCNGGA TCCRAC(20/18)* TCGCGA (9/11)TCGGTC* TCTAGA (10/12)TGA(N)6TCA(12/10) TGATCA TGCGCA TGGCCA (9/11)TGGGTG* (9/11)TGGYTA* TGTACA TTATAA TTCGAA TTTAAA WCCGGW WGTACW YACGTR YGGCCR

Conventional enzyme BamHI EheI (SfoI) NarI SspDI (KasI) BbeI EciI Bsp120I (PspOMI) ApaI KpnI Acc65I (Asp718I) Eco31I (BsaI) Eco91I (BstEII) BshNI (BanI) BseSI (Bme1580I) Hin1I (BsaHI) Eco24I (BanII) Bst1107I (BstZ17I) BfuI (BciVI) SalI BpiI (BbsI) Alw44I (ApaLI) BsgI XmiI (AccI) KspAI (HpaI) MmeI HincII (HindII) Alw21I (BsiHKAI) XapI (ApoI) XceI (NspI) Cfr10I (BsrFI) PsuI (BstYI) HaeII EcoO109I (DraII) Eco105I (SnaBI) TsoI PagI (BspHI) EciI Kpn2I (BspEI) PfoI MmeI Bsp68I (NruI) TaqII XbaI BdaI BclI NsbI (FspI) MlsI (MscI) TaqII TsoI Bsp1407I (BsrGI) AanI (PsiI) Bsp119I (BstBI) DraI BsaWI TatI Ppu21I (BsaAI) CfrI (EaeI) Specificity 53 ACCWGGT (10/15)AC(N)4GTAYC(12/7)* (11/13)CAA(N)5GTGG(12/10)* CACCTGC(4/8)* (6/11)CCAA(N)7TTC(12/7)* (10/12)CCAC(N)5TTG(13/11)* CCCWGGG CCTCAGC(-5/-2)* CGGWCCG (6/11)CRAA(N)6GTC(13/8)* (7/12)GAAC(N)5CTC(13/8)* (7/12)GAAC(N)6TAC(12/7)* (7/12-13)GAAC(N)6TCC(12-13/7)* (4/1)GAAGAGC* (7/12)GAAG(N)6TAC(12/7)* (7/12)GAA(N)7TTGG(11/6)* (8/13)GAC(N)6TTYG(11/6)* (8/13)GAG(N)5GTTC(12/7)* (8/4)GCAGGTG* GCTCTTC(1/4)* GCTGAGG(-5/-2)* (7/12-13)GGA(N)6GTTC(12-13/7)* GGGWCCC (7/12)GRTACNNNNGT(15/10)* (7/12)GTA(N)6CTTC(12/7)* (7/12)GTA(N)6GTTC(12/7)* RGGWCCY Conventional enzyme SexAI BaeI CspCI AarI FastDigest AjuI AjuI CspCI PasI BbvCI CpoI (RsrII) FastDigest RsrII (CpoI) ArsI PpiI PsrI AloI LguI (SapI) FastDigest SapI (LguI) BarI FastDigest AjuI AjuI ArsI PpiI AarI LguI (SapI) FastDigest SapI (LguI) BbvCI AloI SanDI FastDigest SanDI (KflI) BaeI BarI PsrI FastDigest PpuMI (Psp5II) Psp5II (PpuMI) FastDigest enzyme FastDigest SexAI (CsiI) FastDigest enzyme FastDigest BamHI FastDigest EheI
FastDigest Bsp120I FastDigest ApaI FastDigest KpnI FastDigest Acc65I FastDigest Eco31I FastDigest Eco91I FastDigest BanI (BshNI) FastDigest Bme1580I (BseSI) FastDigest BsaHI (Hin1I) FastDigest BstZ17I (Bst1107I) FastDigest SalI FastDigest BbsI (BpiI) FastDigest ApaLI (Alw44I) FastDigest AccI (XmiI) FastDigest HpaI (KspAI) FastDigest HincII FastDigest Alw21I FastDigest XapI FastDigest NspI (XceI) FastDigest BsrFI (Cfr10I) FastDigest PsuI FastDigest HaeII (BfoI) FastDigest EcoO109I FastDigest SnaBI (Eco105I) FastDigest BspHI (PagI) FastDigest Kpn2I FastDigest PfoI FastDigest NruI (RruI) FastDigest XbaI FastDigest BclI FastDigest FspI (NsbI) FastDigest MscI (MlsI)

Specificity 53 ATTTAAAT CCTCGAGG CCTGCAGG CGCCGGCG CGCGCGCG CGTCGACG CRCCGGYG GCCCGGGC GCGATCGC GCGGCCGC GGCCGGCC GGCCNNNNNGGCC GGCGCGCC GTTTAAAC RTGCGCAY TTAATTAA VCTCGAGB FastDigest enzyme FastDigest SwaI (SmiI) FastDigest SbfI (SdaI) FastDigest MreI FastDigest MauBI Conventional enzyme SmiI (SwaI) AbsI SdaI (SbfI) MreI (Sse232I) MauBI SgrDI SgrAI SrfI SfaAI (AsiSI) NotI FseI SfiI SgsI (AscI) MssI (PmeI) FspAI PacI PspXI
FastDigest AsiSI (SfaAI) FastDigest NotI FastDigest SfiI FastDigest AscI (SgsI) FastDigest MssI FastDigest FspAI FastDigest PacI
FastDigest Bsp1407I FastDigest PsiI (AanI) FastDigest Bsp119I FastDigest DraI FastDigest TatI FastDigest BsaAI (Ppu21I)
Single letter code

R = G or A; Y = C or T; W = A or T; K = G or T; S = C or G; H = A, C or T; B = C, G or T; D = A, G or T; N = G, A, T or C; M = A or C; V = A, C or G.
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Commercial Restriction Enzymes Generating 5-protruding Ends

Table 1.33. Commercial restriction enzymes generating 5-protruding ends. Recognition sequence AACGTT AAGCTT AATT ACATGT ACCGGT ACCTGC(4/8)* ACCWGGT ACGCGT ACGGC(12/14)* ACGT ACRYGT ACTAGT AGATCT ATCGAT ATTAAT CAATTG CACCTGC(4/8)* CACGAG(-5/-1)* CAGCAG(25/27)* CATATG (13/9)CATCC* (14/10)CATCGC* CATG CATG CCATC(4/5)* CCATGG CCCAGC(-5/-1)* CCCGC(4/6)* CCCGGG CCCWGGG CCGC(-3/-1)* CCGG CCNGG CCNGG CCNNGG CCRYGG CCSGG CCTAGG CCTCAGC(-5/-2)* CCTCGAGG CCTNAGC(-5/-2)* CCTNAGG CCTNNNNNAGG CCWGG CCWGG CCWWGG CGCCGGCG CGCGCGCG CGGCCG CGGWCCG CGTACG 5mCNNG(9/13) CGTCGACG CGTCTC(1/5)* CRCCGGYG FastDigest enzyme FastDigest AclI (Psp1406I) FastDigest HindIII FastDigest Tsp509I (TasI) FastDigest AgeI (BshTI) FastDigest BspMI (BveI) FastDigest SexAI (CsiI) FastDigest MluI Conventional enzyme Psp1406I (AclI) HindIII TasI (Tsp509I) PscI (PciI) BshTI (AgeI) BveI (BspMI) SexAI MluI BceAI MaeII AflIII BcuI (SpeI) BglII Bsu15I (ClaI) VspI (AseI) MunI (MfeI) AarI BauI (BssSI) EcoP15I NdeI FokI BtgZI FatI CviAII BccI NcoI BseYI SmuI (FauI) Cfr9I (XmaI) PasI SsiI (AciI) HpaII MspI (HpaII) StyD4I Bme1390I (ScrFI) BseDI (BsaJI) BtgI BcnI (NciI) XmaJI (AvrII) BbvCI AbsI Bpu10I Eco81I (Bsu36I) XagI (EcoNI) EcoRII MvaI (BstNI) Eco130I (StyI) MreI (Sse232I) MauBI Eco52I (EagI) CpoI (RsrII) Pfl23II (BsiWI) SgeI SgrDI Esp3I (BsmBI) SgrAI 1 nt 5-protruding end (5 3) 2 nt 3 nt 4 nt CG AGCT AATT CATG CCGG NNNN CGCG NN CG CRYG CTAG GATC CG TA AATT NNNN ACGA NN TA NNNN NNNN CATG AT N CATG CCAG NN CCGG CWG CG CG CCNGG N CNNG CRYG S CTAG TCA TCGA TNA TNA N CCWGG W CWWG CCGG CGCG GGCC GWC GTAC NNNN TCGA NNNN CCGG
1
5 nt
CCWGG
FastDigest SpeI (BcuI) FastDigest BglII FastDigest ClaI (Bsu15I) FastDigest AseI (VspI) FastDigest MfeI (MunI)
FastDigest NdeI FastDigest FokI
FastDigest NcoI
FastDigest AciI (SsiI) FastDigest HpaII FastDigest MspI FastDigest ScrFI (Bme1390I) FastDigest BsaJI (BseDI) FastDigest NciI (BcnI) FastDigest AvrII (XmaJI)
FastDigest Bpu10I FastDigest Bsu36I (Eco81I) FastDigest EcoNI (XagI) FastDigest MvaI FastDigest StyI (Eco130I) FastDigest MreI FastDigest MauBI FastDigest EagI (Eco52I) FastDigest RsrII (CpoI) FastDigest BsiWI (Pfl23II)
FastDigest BsmBI (Esp3I)
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Table 1.33.Commercial restriction enzymes generating 5-protruding ends.
1
Recognition sequence CTAG CTCGAG CTCGTG(-5/-1)* CTCTTC(1/4)* (27/25)CTGCTG* CTNAG CTRYAG CTTAAG CTYRAG CYCGRG CYCGRG GAAGAC(2/6)* (4/1)GAAGAG* (4/1)GAAGAGC* GAATTC GACGC(5/10)* GACNNNGTC (5/4)GACTC* (5/1)GAGAC* (5/1)GAGACC* (5/1)GAGACG* GAGTC(4/5)* GANTC GATC (5/4)GATCC* (9/5)GATGC* (5/4)GATGG* GAWTC GCAGC(8/12)* (8/4)GCAGGT* (8/4)GCAGGTG* GCATC(5/9)* GCCGGC (14/12)GCCGT* GCGATG(10/14)* GCGC GCGCGC GCGG(-3/-1)* GCGGCCGC (6/4)GCGGG* (10/5)GCGTC* GCNGC GCTAGC GCTCTTC(1/4)* GCTGAGG(-5/-2)* (12/8)GCTGC* GCTGGG(-5/-1)* GCTNAGC GCTNAGG(-5/-2)* GCWGC GGATC(4/5)* GGATCC GGATG(9/13)* GGCGCC GGCGCC GGCGCGCC GGGAC(10/14)*
FastDigest enzyme FastDigest BfaI (FspBI) FastDigest XhoI FastDigest EarI (Eam1104I) FastDigest DdeI (HpyF3I) FastDigest SfcI (BfmI) FastDigest AflII (BspTI) FastDigest AvaI (Eco88I) FastDigest BbsI (BpiI) FastDigest EarI (Eam1104I) FastDigest SapI (LguI) FastDigest EcoRI FastDigest HgaI (CseI) FastDigest PsyI FastDigest Alw26I FastDigest Eco31I FastDigest BsmBI (Esp3I) FastDigest HinfI FastDigest MboI FastDigest Sau3AI (Bsp143I) FastDigest SfaNI (BmsI) FastDigest TfiI (PfeI) FastDigest BbvI (Lsp1109I) FastDigest BseXI (BbvI) FastDigest BspMI (BveI) FastDigest SfaNI (BmsI)
FastDigest HinP1I (Hin6I) FastDigest BssHII (PteI) FastDigest AciI (SsiI) FastDigest NotI FastDigest HgaI (CseI) FastDigest Fnu4HI (SatI) FastDigest NheI FastDigest SapI (LguI) FastDigest BbvI (Lsp1109I) FastDigest BseXI (BbvI) FastDigest BlpI (Bpu1102I) FastDigest Bpu10I
FastDigest BamHI FastDigest FokI
FastDigest AscI (SgsI) FastDigest BsmFI (FaqI)
Conventional enzyme FspBI (BfaI) XhoI BauI (BssSI) Eam1104I (EarI) EcoP15I HpyF3I (DdeI) BfmI (SfcI) BspTI (AflII) SmoI (SmlI) Eco88I (AvaI) BmeT110I BpiI (BbsI) Eam1104I (EarI) LguI (SapI) EcoRI CseI (HgaI) PsyI (Tth111I) PleI Alw26I (BsmAI) Eco31I (BsaI) Esp3I (BsmBI) PleI HinfI MboI Bsp143I (Sau3AI) BspPI (AlwI) LweI (SfaNI) BccI PfeI (TfiI) Lsp1109I (BbvI) BseXI (BbvI) BveI (BspMI) AarI LweI (SfaNI) NgoMIV BceAI BtgZI Hin6I (HinP1I) PauI (BssHII) SsiI (AciI) NotI SmuI (FauI) CseI (HgaI) SatI (Fnu4HI) NheI LguI (SapI) BbvCI Lsp1109I (BbvI) BseXI (BbvI) BseYI Bpu1102I (BlpI) Bpu10I TseI BspPI (AlwI) BamHI FokI NarI SspDI (KasI) SgsI (AscI) FaqI (BsmFI)
1 nt
5-protruding end (5 3) 2 nt 3 nt 4 nt TA TCGA TCGT NNN NN TNA TRYA TTAA TYRA YCGR CG NNNN NNN NNN AATT
5 nt
NNNNN N N NNNN NNNN NNNN N ANT GATC N NNNN N AWT NNNN NNNN NNNN NNNN CCGG NN NNNN CG CGCG CG GGCC NN NNNNN N CTAG NNN TGA NNNN CTGG TNA TNA CWG N GATC NNNN CG GCGC CGCG NNNN
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Table 1.33.Commercial restriction enzymes generating 5-protruding ends. Recognition sequence GGGCCC GGGWCCC GGNCC GGNCC GGTACC GGTCTC(1/5)* GGTNACC GGWCC GGYRCC GRCGYC GTAC (14/10)GTCCC* GTCGAC GTCTC(1/5)* (6/2)GTCTTC* GTGCAC GTMKAC GTNAC GTSAC RAATTY RCCGGY RGATCY RGGNCCY RGGWCCY TCATGA TCCGGA TCCNGGA TCGA TCNNGA TCTAGA TGATCA TGTACA TTAA TTCGAA VCTCGAGB WCCGGW WGTACW YGGCCR FastDigest enzyme FastDigest Bsp120I FastDigest SanDI (KflI) FastDigest Sau96I (Cfr13I) FastDigest Acc65I FastDigest Eco31I FastDigest Eco91I FastDigest AvaII (Eco47I) FastDigest BanI (BshNI) FastDigest BsaHI (Hin1I) FastDigest Csp6I FastDigest BsmFI (FaqI) FastDigest SalI FastDigest Alw26I FastDigest BbsI (BpiI) FastDigest ApaLI (Alw44I) FastDigest AccI (XmiI) FastDigest NmuCI FastDigest XapI FastDigest BsrFI (Cfr10I) FastDigest PsuI FastDigest EcoO109I FastDigest PpuMI (Psp5II) FastDigest BspHI (PagI) FastDigest Kpn2I FastDigest PfoI FastDigest TaqI FastDigest XbaI FastDigest BclI FastDigest Bsp1407I FastDigest MseI (SaqAI) FastDigest Tru1I FastDigest Bsp119I
Conventional enzyme 1 nt Bsp120I (PspOMI) SanDI Cfr13I (Sau96I) BmgT120I N Acc65I (Asp718I) Eco31I (BsaI) Eco91I (BstEII) Eco47I (AvaII) BshNI (BanI) Hin1I (BsaHI) Csp6I (CviQI) FaqI (BsmFI) SalI Alw26I (BsmAI) BpiI (BbsI) Alw44I (ApaLI) XmiI (AccI) MaeIII NmuCI (Tsp45I) XapI (ApoI) Cfr10I (BsrFI) PsuI (BstYI) EcoO109I (DraII) Psp5II (PpuMI) PagI (BspHI) Kpn2I (BspEI) PfoI TaqI Hpy188III XbaI BclI Bsp1407I (BsrGI) Tru1I (MseI) Bsp119I (BstBI) PspXI BsaWI TatI CfrI (EaeI)
5-protruding end (5 3) 2 nt 3 nt 4 nt GGCC GWC GNC GTAC NNNN
5 nt
GTNAC GWC GYRC CG TA NNNN TCGA NNNN NNNN TGCA MK GTNAC GTSAC AATT CCGG GATC GNC GWC CATG CCGG CCNGG CG NN CTAG GATC GTAC TA CG TCGA CCGG GTAC GGCC
FastDigest TatI
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Commercial Restriction Enzymes Generating 3-protruding Ends

Table 1.34. Commercial restriction enzymes generating 3-protruding ends. Recognition sequence ACGGA(11/9)* ACGT ACNGT ACTGG(1/-1)* ACTGGG(5/4)* ASST ATGAA(11/9)* ATGCAT CACCCA(11/9)* CACNNNGTG (0/2)CACTGC* CAGNNNCTG CASTG(2/-7) (0/2)CATCC* CATG (0/2)CATTGC* (-1/1)CCAGT* CCANNNNNNNNNTGG CCANNNNNNTGG CCANNNNNTGG CCCAGC(-1/-5)* (4/5)CCCAGT* CCGCGG CCNNNNNNNGG CCTC(7/6)* CCTGCAGG CCTTC(6/5)* CGATCG CGRYCG CGWCG (14/16)CTCAAG* CTCAG(9/7)* CTCAG(10/8)* (14/16)CTCCAG* (8/10)CTCCTC* (19/21)CTCGGC* CTGAAG(16/14)* (7/9)CTGAG* (8/10)CTGAG* (14/16)CTGCAC* CTGCAG CTGGAG(16/14)* CTGRAG(16/14)* (14/16)CTTCAG* CTTGAG(16/14)* (14/16)CTYCAG* GAAGA(8/7)* (5/6)GAAGG* GAATGC(1/-1)* GACCGA(11/9)* GACGTC GACNNNNNGTC GACNNNNNNGTC GAGCTC (6/7)GAGG* GAGGAG(10/8)* GATC GCAATG(2/0)* GCAGTG(2/0)* Conventional enzyme TspGWI FastDigest TaiI TaiI (MaeII) FastDigest TaaI TaaI (HpyCH4III) FastDigest BseNI BseNI (BsrI) BfiI (BmrI) SetI TspDTI FastDigest NsiI (Mph1103I) Mph1103I (NsiI) TaqII FastDigest DraIII (AdeI) AdeI (DraIII) BtsI FastDigest AlwNI (CaiI) CaiI (AlwNI) FastDigest TspRI (TscAI) TscAI (TspRI) FastDigest BseGI BseGI (BtsCI) FastDigest NlaIII (Hin1II) Hin1II (NlaIII) FastDigest BsrDI (BseMI) BseMI (BsrDI) FastDigest BseNI BseNI (BsrI) XcmI FastDigest BstXI BstXI FastDigest PflMI (Van91I) Van91I (PflMI) FastDigest PspFI GsaI BfiI (BmrI) Cfr42I (SacII) FastDigest BslI (BseLI) BseLI (BslI) FastDigest MnlI MnlI FastDigest SbfI (SdaI) SdaI (SbfI) HpyAV FastDigest PvuI PvuI FastDigest BsiEI (Bsh1285I) Bsh1285I (BsiEI) Hpy99I BpuEI BspCNI FastDigest BspCNI (BseMII) BseMII (BspCNI) FastDigest BpmI (GsuI) GsuI (BpmI) BseRI NmeAIII FastDigest AcuI (Eco57I) Eco57I (AcuI) BspCNI FastDigest BspCNI (BseMII) BseMII (BspCNI) BsgI FastDigest PstI PstI FastDigest BpmI (GsuI) GsuI (BpmI) Eco57MI FastDigest AcuI (Eco57I) Eco57I (AcuI) BpuEI Eco57MI FastDigest MboII MboII HpyAV FastDigest Mva1269I Mva1269I (BsmI) TaqII FastDigest AatII AatII FastDigest Eam1105I Eam1105I (AhdI) FastDigest DrdI (AasI) AasI (DrdI) FastDigest SacI SacI FastDigest MnlI MnlI BseRI BstKTI FastDigest BsrDI (BseMI) BseMI (BsrDI) BtsI FastDigest enzyme 1 nt 2 nt NN 3-protruding end (5 3) 3 nt 4 nt 5 nt ACGT N GN N ASST NN TGCA NN NNN NN NNN NNCASTGNN NN CATG NN NC N NNNN NNN CCAG N GC NNN N TGCA N AT RY CGWCG NN NN NN NN NN NN NN NN NN NN TGCA NN NN NN NN NN N N CN NN ACGT N NN AGCT N NN AT NN NN
9 nt
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Table 1.34.Commercial restriction enzymes generating 3-protruding ends. Recognition sequence GCANNNNNTGC GCATGC (-1/1)GCATTC* GCCGAG(21/19)* GCCNNNNNGGC GCGATCGC GCGC GCNNNNNNNGC GCSGC GCTAGC GCTGGG(-1/-5)* GDGCHC (5/6)GGATAC* GGATG(2/0)* GGCCGGCC GGCCNNNNNGGCC GGCGCC GGCGGA(11/9)* GGGCCC GGTACC GGTGA(8/7)* GKGCMC GRGCYC GTATCC(6/5)* GTGCAG(16/14)* (18/20)GTYGGA* GWGCWC RCATGY RGCGCY TARCCA(11/9)* (7/8)TCACC* (9/11)TCCGCC* (9/11)TCCGT* TCCRAC(20/18)* (9/11)TCGGTC* TCNGA (7/8)TCTTC* (9/11)TGGGTG* (9/11)TGGYTA* TTAATTAA (9/11)TTCAT* TTSAA FastDigest enzyme FastDigest SphI (PaeI) FastDigest Mva1269I FastDigest BglI FastDigest AsiSI (SfaAI) FastDigest HhaI FastDigest HpyF10VI FastDigest TauI FastDigest BmtI (BspOI) FastDigest PspFI FastDigest Bsp1286I (SduI) FastDigest BseGI FastDigest SfiI Conventional enzyme 1 nt BstAPI PaeI (SphI) Mva1269I (BsmI) NmeAIII BglI SfaAI (AsiSI) HhaI HpyF10VI (MwoI) TauI BspOI (BmtI) GsaI SduI (Bsp1286I) BfuI (BciVI) N BseGI (BtsCI) FseI SfiI BbeI EciI ApaI KpnI HphI N BseSI (Bme1580I) Eco24I (BanII) BfuI (BciVI) BsgI MmeI Alw21I (BsiHKAI) XceI (NspI) HaeII TsoI HphI EciI TspGWI MmeI TaqII Hpy188I MboII TaqII TsoI PacI TspDTI AgsI N NN NN WGCW CATG GCGC NN N NN NN NN NN N N NN NN AT NN S 3-protruding end (5 3) 2 nt 3 nt 4 nt 5 nt NNN CATG NG NN NNN AT CG NNN CSG CTAG CTGG DGCH NN CCGG NNN GCGC NN GGCC GTAC KGCM RGCY 9 nt
FastDigest ApaI FastDigest KpnI FastDigest Bme1580I (BseSI)
FastDigest Alw21I FastDigest NspI (XceI) FastDigest HaeII (BfoI)
FastDigest MboII
FastDigest PacI
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Commercial Restriction Enzymes Generating Blunt Ends

Table 1.35. Commercial restriction enzymes generating blunt ends. Reccognition Conventional FastDigest sequence enzyme enzyme SspI FastDigest SspI AATATT AGCGCT FastDigest AfeI (Eco47III) Eco47III (AfeI) FastDigest AluI AluI AGCT AGGCCT FastDigest StuI (Eco147I) Eco147I (StuI) FastDigest ScaI ScaI AGTACT ATTTAAAT FastDigest SwaI (SmiI) SmiI (SwaI) CACGTC(-3/-3)* AjiI (BmgBI) CACGTG FastDigest PmlI (Eco72I) Eco72I (PmlI) CACNNNNGTG FastDigest AleI (OliI) OliI (AleI) FastDigest PvuII PvuII CAGCTG CAYNNNNRTG FastDigest MslI (RseI) RseI (MslI) FastDigest SmaI SmaI CCCGGG CCGCTC(-3/-3)* FastDigest BsrBI (MbiI) MbiI (BsrBI) FastDigest Bsh1236I CGCG Bsh1236I (BstUI) CMGCKG MspA1I FastDigest PdmI GAANNNNTTC PdmI (XmnI) GACGTC ZraI GACGTG(-3/-3)* AjiI (BmgBI) GACNNNNGTC FastDigest PshAI (BoxI) BoxI (PshAI) (5/5)GACTC* FastDigest MlyI (SchI) SchI (MlyI) GAGCGG(-3/-3)* FastDigest BsrBI (MbiI) MbiI (BsrBI) FastDigest Ecl136II GAGCTC Ecl136II (EcoICRI) GAGTC(5/5)* FastDigest MlyI (SchI) SchI (MlyI) GATATC FastDigest EcoRV (Eco32I) Eco32I (EcoRV) FastDigest DpnI DpnI GATC GATNNNNATC FastDigest BsaBI (BseJI) BseJI (BsaBI) GCCCGGGC SrfI GCCGGC FastDigest NaeI (PdiI) PdiI (NaeI) GCGC GlaI GCNNGC Cac8I GGCC FastDigest HaeIII (BsuRI) BsuRI (HaeIII) FastDigest EheI GGCGCC EheI (SfoI) GGNNCC FastDigest NlaIV (BspLI) BspLI (NlaIV) FastDigest RsaI RsaI GTAC GTATAC FastDigest BstZ17I (Bst1107I) Bst1107I (BstZ17I) FastDigest Hpy8I GTNNAC Hpy8I (MjaIV) GTTAAC FastDigest HpaI (KspAI) KspAI (HpaI) FastDigest MssI GTTTAAAC MssI (PmeI) FastDigest HincII GTYRAC HincII (HindII) RGCY CviJI FastDigest FspAI FspAI RTGCGCAY TACGTA FastDigest SnaBI (Eco105I) Eco105I (SnaBI) TCGCGA FastDigest NruI (RruI) Bsp68I (NruI) TGCA HpyCH4V TGCGCA FastDigest FspI (NsbI) NsbI (FspI) TGGCCA FastDigest MscI (MlsI) MlsI (MscI) TTATAA FastDigest PsiI (AanI) AanI (PsiI) FastDigest DraI DraI TTTAAA YACGTR FastDigest BsaAI (Ppu21I) Ppu21I (BsaAI) YATR FaiI
Commercial Restriction Enzymes Cleaving DNA on the Both Sides of their Recognition Sequence
Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence. Recognition sequence (8/13)AAG(N)5CTT(13/8) (9/12)AC(N)5CTCC(10/7) (10/15)AC(N)4GTAYC(12/7) (11/13)CAA(N)5GTGG(12/10) (8/13)CAC(N)6TCC(12/7) (6/11)CCAA(N)7TTC(12/7) (10/12)CCAC(N)5TTG(13/11) (10/12)CGA(N)6TGC(12/10) (6/11)CRAA(N)6GTC(13/8) (7/12)GAA(N)7TTGG(11/6) (7/12)GAAC(N)5CTC(13/8) (7/12)GAAC(N)6TAC(12/7) (7/12-13)GAAC(N)6TCC(12-13/7) (7/12)GAAG(N)6TAC(12/7) (8/13-14)GAB(N)5RTC(13-14/8) (8/13)GAC(N)6TTYG(11/6) (8/13)GAG(N)5CTC(13/8) (8/13)GAG(N)5GTTC(12/7) (8/13-14)GAY(N)5VTC(13-14/8) (10/12)GCA(N)6TCG(12/10) (10/12)GCA(N)6TGC(12/10) (7/12)GGA(N)6GTG(13/8) (7/12-13)GGA(N)6GTTC(12-13/7) (7/10)GGAG(N)5GT(12/9) (7/12)GRTAC(N)4GT(15/10) (7/12)GTA(N)6CTTC(12/7) (7/12)GTA(N)6GTTC(12/7) (10/12)TGA(N)6TCA(12/10) FastDigest enzyme Conventional enzyme FalI BsaXI BaeI CspCI TstI AjuI CspCI BcgI ArsI AjuI PpiI PsrI AloI BarI Hin4I ArsI BplI PpiI Hin4I BcgI AlfI TstI AloI BsaXI BaeI BarI PsrI BdaI
FastDigest AjuI
FastDigest AjuI
FastDigest BplI
Single letter code

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Troubleshooting Guide
Digestion problem 1 Incomplete digestion, no digestion 2 Unexpected cleavage pattern 3 Diffuse DNA bands on gel
Assess enzyme activity with control DNA
2.1 Star activity
3.1 Gel shift
1.1 Inactive enzyme
Active enzyme
2.2 Contamination with another RE
3.2 Contaminated reagents
1.2 Suboptimal reaction conditions
1.3 Unsuitable or contaminated DNA
1.4 Water contains impurities
2.3 Contamination with another DNA
1.2.1 Suboptimal digestion protocol
Evaluate inhibition by template DNA solution
2.4 Incomplete DNA digestion
1.2.2 Improper enzyme dilution
Inhibition
No inhibition
2.5 Gel shift
1.2.3 Improper reaction assembly
1.3.1 Contaminants in DNA solution
1.3.2 Absence of recognition sites
2.6 Unexpected sites in template DNA
1.2.4 Excess glycerol
1.3.3 Cleavage blocked by methylation
1.2.5 Suboptimal DNA concentration
1.3.4 Structure of DNA substrate
1.3.4.1 Supercoiled plasmid DNA
1.3.4.2 Proximity of site to DNA ends
1.3.4.3 At least two sites required
1.3.4.4 Site preference
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Table 1.37. Troubleshooting guide for DNA digestion.
1
Problem
Possible cause and recommended solution Assess enzyme activity The restriction enzyme may lose activity due to improper storage or handling. Perform a digestion reaction with 1g of a standard control DNA, e.g. Lambda DNA (dam, dcm) (#SD0021). 1.1. Inactive enzyme. If the enzyme does not cut the control DNA: Check the expiration date. Verify that the enzyme has been stored at -20C. Check the temperature of your freezer. Do not allow the temperature go below -20C as the enzyme may freeze and multiple freeze thaw cycles (more than 3 cycles) may result in reduced enzyme activity. 1.2. Suboptimal reaction conditions. 1.2.1. Suboptimal digestion protocol. Follow digestion protocol specified for the restriction enzyme and type of substrate DNA. All FastDigest enzymes are experimentally tested on Lambda DNA (or other control substrate DNA), plasmid and genomic DNA as well as PCR products. Please refer to Table 1.3 on p.71 for specific recommendations. For FastDigest enzymes use FastDigest or FastDigest Green Buffer. All FastDigest enzymes are 100% active in these buffers. For conventional restriction enzymes use the recommended reaction buffer supplied with the enzyme. For double digestions follow the recommendations of the DoubleDigest engine at www.fermentas.com/doubledigest. Use additives where required. Perform the reaction at the optimal temperature specified for the restriction enzyme. For double digestions with enzymes requiring different incubation temperatures perform sequential DNA cleavage: complete the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second enzyme cleavage. Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation; the increase in salt concentration may reduce enzyme activity. For thermophilic enzymes use a heat block with a hot bonnet, e.g. a PCR cycler. 1.2.2. Improper dilution of conventional enzyme. Dilute conventional restriction enzymes with Dilution Buffer for Restriction Enzymes (#B19). Restriction enzymes diluted with this buffer are stable for at least 3-4 weeks at -20C. Never dilute enzymes in water or 10Xreaction buffer. Never dilute enzymes in 1Xreaction buffer in the absence of DNA. Use the recommended amount of FastDigest enzymes, which are experimentally tested on four different DNA substrates (see Table 1.3 on p.71). 1.2.3. Improper reaction assembly. The restriction enzyme should always be the last component added to the reaction mixture. The restriction enzyme may be inactivated if added directly to a 10Xreaction buffer. 1.2.4. Excess glycerol in the reaction mixture. The glycerol concentration in the reaction mixture should not exceed 5%. Thus, the volume of the restriction enzyme added to the mixture should not exceed 1/10 of the total reaction volume. Enzymes sensitive to high glycerol concentration include: Alw21I, BpiI, Bsp68I, BspTI, Eco32I, Eco91I, EcoRI, Hin6I, HinfI, Mph1103I, Mva1269I and NcoI. 1.2.5. Suboptimal DNA concentration. The optimal range of DNA concentration in the reaction mixture is 0.02-0.1g/l. 1.3. Unsuitable DNA template or contaminated DNA solution. If the enzyme is active in the control digest, assay the substrate DNA solution for inhibitory contaminants in a mixing experiment with control template, e.g. Lambda DNA (dam, dcm) (#SD0021). Perform a control digest with two templates, control and sample, in one reaction mixture. Do not exceed the optimal DNA concentration in the reaction mixture (0.02-0.1g/l). The sample template is contaminated if neither the control DNA nor sample DNA template is digested (see 1.3.1). The sample template is not contaminated if the control DNA template is digested but the sample template is not. Poor digestion of the experimental template is caused by errors in the DNA sequence (see 1.3.2), methylation effects (see 1.3.3) or structure of the DNA substrate (see 1.3.4).
1. Incomplete digestion or no digestion
Note

Always ensure that the control DNA contains a recognition site for the enzyme present in the reaction. For example, there is no NotI recognition site in lambda DNA. (continued on next page)
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Table 1.37.Troubleshooting guide for DNA digestion.
Problem
Possible cause and recommended solution 1.3.1. Contaminants in the DNA solution. Template DNA may contain residual SDS, EDTA, proteins, salts or nucleases. Repurify the template using a GeneJET PCR Purification Kit (#K0701) or by phenol/chloroform extraction and ethanol precipitation (seep.358). DNA A260/280 ratio should be 1.8-2.0. To remove EDTA and salts, wash the pellet with 70% cold ethanol. For reliable and reproducible plasmid miniprep purity, use the GeneJET Plasmid Miniprep Kit (#K0502). For digestion of unpurified PCR products, dilute DNA at least 3fold in the recommended 1Xrestriction enzyme buffer see protocols on p.70 or p.160. If the template DNA has been purified using silica or resin suspensions, remove all remaining particles by centrifugation for 10min at 10,000 rpm and ensure that no resin is carried over while transferring the DNA solution into a new tube. 1.3.2. The substrate DNA does not contain a recognition sequence for the restriction enzyme. Re-check the DNA sequence and cloning strategy. Determine if the restriction enzyme selected requires more than one site per target DNA for 100% activity (see also 1.3.4.3). Check literature for known site preferences for the restriction enzyme (see also 1.3.4.4). If the recognition sequence was introduced by PCR primers, verify that the primer sequence contains the recognition site. 1.3.3. Methylation effects. Restriction enzyme may be inhibited by methylation of the recognition site. Identify which type of DNA methylation can occur on the recognition site and determine if the methylation impairs or blocks DNA digestion with the enzyme. See Digestion of Methylated DNA on p.175 and use Tables 1.13 and 1.20 on pp.176-181. If methylation impairs or blocks DNA cleavage: Propagate your plasmid in an E.coli dam, dcm strain (the E.coli GM2163 dam, dcm strain; #M0099, is available upon request with the purchase of any Fermentas product), Use the REsearch engine at www.fermentas.com/research or check the Fermentas catalog for the availability of a restriction enzyme isoschizomer not sensitive to DNA methylation. Using of restriction enzyme which requires a methylated recognition sequence (DpnI or SgeI) for digestion of unmethylated DNA will result in no DNA cleavage. Propagate your plasmid in E.coli dam+ or dcm+ strains (please refer to p.486 for genotype information of some common E.coli strains) to get DNA with methylated DpnI or SgeI recognition sequences. Alternatively, in the case of DpnI, the neoschizomers Bsp143I or MboI can be used to digest non-methylated DpnI recognition sites.
Note
When PCR is carried out with standard dNTPs and non-methylated primers the resulting DNA product is not methylated. 1.3.4. Structure of substrate DNA. 1.3.4.1. Supercoiled plasmid DNA. Use FastDigest enzymes which are qualified for supercoiled DNA and provided with specific recommendations for each enzyme (see Table 1.3 on p.71). For some conventional restriction enzymes, additional units are required to completely digest supercoiled plasmids (e.g. 5-10 u (1l ) of restriction enzyme per 1g of DNA). Check notes in the catalog description of the enzyme or refer to the Certificate of Analysis. 1.3.4.2. Proximity of the recognition sequence to the DNA ends. Some restriction enzymes cleave DNA poorly if the recognition site is too close to the end of the DNA molecule. For FastDigest enzymes refer to Table 1.3 on p.71 or the product description to determine the effectiveness of restriction enzyme cleavage at the ends of DNA. For conventional restriction enzymes refer to Tables 1.10 (p.171) and 1.11 (p.172). 1.3.4.3. Restriction enzyme requires at least two sites per DNA molecule for optimal activity. Some restriction enzymes such as AarI, BveI, Cfr10I, Cfr42I, Eco57I, EcoRII, LweI, SfiI require at least two target sites per DNA molecule for efficient cleavage (for more details see Site Preferences by Restriction Enzymes on p.173). If there is only one recognition site per DNA molecule, add a DNA oligonucleotide containing the recognition site. 1.3.4.4. Site Preferences by Restriction Enzymes. The DNA sequence surrounding the recognition site may influence the efficiency of digestion. Some DNA sites are cleaved slowly or not cleaved at all (for more details see p.173) due to the surrounding sequence. Use additional units (5-10 u) of conventional restriction enzyme per 1g of DNA or determine if an isoschizomer has superior cleavage efficiency (see Table 1.25 on p.207 or REsearch engine at www.fermentas.com/research).
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Table 1.37.Troubleshooting guide for DNA digestion.
1
Problem
Possible cause and recommended solution 1.4. Water contains impurities. Compare your results using commercially available nuclease free, molecular biology grade water, e.g. Water, nuclease-free (#R0581). Check the quality of the water used in your lab. Check the pHand conductivity of water. The pHof high quality water should be 5.5-6.0 with a resistance of >18 M. Centrifuge (10 min, 10,000 rpm) 1 ml of water and check if there is a visible pellet. Determine if water contains nucleases or bacterial contamination (see 3.2 for control reactions). 2.1. Star activity (relaxed specificity) of restriction enzyme (see p.174 for more details). Use FastDigest restriction enzymes. For these enzymes the incubation time without star activity is determined. Reduce the amount of conventional restriction enzyme (use no more than 10u). Use the recommended reaction buffer. Ensure that the glycerol concentration in the reaction mixture does not exceed 5%. Reduce the incubation time. For FastDigest enzymes refer to Table 1.3 on p.71 for maximum incubation times. Ensure the volume of the reaction mixture was not reduced due to evaporation during incubation. The resulting increase in glycerol concentration may cause star activity. 2.2. Contamination with another restriction enzyme. The restriction enzyme or buffer may be contaminated with another restriction enzyme due to improper handling. Use a new tube of enzyme and/or buffer. 2.3. Contamination with another substrate DNA. The sample DNA may contain a mixture of two or more different DNAs. Prepare new sample of DNA. For plasmid DNA preparation pick one isolated colony of recombinant E.coli grow cells and purify plasmid with GeneJET Plasmid Miniprep Kit (#K0503). For PCR products: check the product purity on an agarose gel. If necessary, purify the PCR product prior to digestion with GeneJET Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513). 2.4. Incomplete DNA digestion (see 1). 2.5. Gel shift (see 3.1). 2.6. Unexpected recognition sites in template DNA. Newly generated target sites in constructed DNA may be overlooked. Re-check your DNA sequence and cloning strategy. Refer to Tables 1.21, 1.22, 1.23 or 1.24 for Newly Generated Recognition Sequences (pp.182-206) to identify all the cleavage sites present in the substrate DNA. 3.1. Gel shift. Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion products. FastDigest Restriction Enzymes BspMI (BveI), HgaI (CseI), AcuI (Eco57I), FokI, MboII, TauI, TspRI (TscAI) and conventional restriction enzymes AarI, AloI, BdaI, BseXI, BveI, CseI, Eco57I, Eco57MI, EcoRII, FaqI, GsuI, Lsp1109I, LweI, MboII, MnlI, SchI, TauI, TscAI, TsoI and TstI are particularly prone to remaining bound to the substrate DNA. This will result in a band or smear above the expected band (see picture below). Heat the digested DNA for 10 min. at 65C in the presence of 6XDNA Loading Dye & SDS Solution (#R1151) or 0.2% SDS prior to electrophoresis.
2. Unexpected cleavage pattern
3. Diffused DNA bands
M GeneRuler DNA Ladder Mix (#SM0331). 1 0.5g DNA prepared for loading with 6XDNA Loading Dye (#R0611). 2 0.5g DNA prepared for loading with 6XDNA Loading Dye & SDS Solution (#R1151). 3 0.5g DNA digested with TsoI (#ER1991), probe prepared for loading with 6XDNA Loading Dye (#R0611). 4 0.5g DNA digested with TsoI, probe prepared for loading with 6XDNA Loading Dye & SDS Solution (#R1151).
3.2. Contaminated reagents. Any restriction digestion reaction components may become contaminated with nucleases due to improper handling or storage. Nuclease contamination causes DNA degradation, which appears as diffused DNA bands on a gel. Perform four control reactions in order to check for the nuclease contamination: I without restriction enzyme, II with a new vial of buffer, III without restriction enzyme, with a new vial of buffer, IV with commercially available water e.g. Water, nuclease-free (#R0581). Contaminated sample DNA (diffused bands in all controls). Prepare new DNA sample (re-purify DNA). Contaminated enzyme (diffused bands in controls 2 and 4). The enzyme may become contaminated due to improper handling. Use a new vial of enzyme. Contaminated buffer (diffused bands in controls 1 and 4). Bacterial contamination of the reaction buffer will cause DNA degradation. Use a new vial of buffer. Store all buffers at -20C. Contamination of both enzyme & buffer (diffused bands in controls 1, 2 and 4). Follow the recommendations given above. Contaminated water (diffused bands in controls 1, 2 and 3). Bacterial or DNase contamination in improperly handled water will cause DNA degradation. Use commercially available nuclease-free molecular biology grade water (e.g. #R0581).
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01 Fastdigest Conventional Restriction Enzymes

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01 Fastdigest Conventional Restriction Enzymes

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FastDigest and Conventional Restriction Enzymes...................................................

FastDigest & CONVENTIONAL RESTRICTION ENZYMES

FastDigest and Conventional Restriction Enzymes

Labeled Oligonucleotide (LO) Test

Labeled Oligonucleotide Test

Degradation due to contamination with non-specific nucleases

& CONVENTIONAL RESTRICTION ENZYMES

For patent and license information see p.557

Bulk quantities and custom formulations available upon request

Single letter code

FastDigest Restriction Enzymes

Universal FastDigest Buffer

FastDigest Green Buffer

For patent and license information see p.557

Bulk quantities and custom formulations available upon request

Activity and Quality Control Assays

Ligation and Recleavage Assay

Blue/White (B/W) Cloning Assay

Quality Control Labeled Oligonucleotide (LO) Test

Prolonged Incubation / Star Activity Assay

Storage and Shipping

(continued on next page)

For patent and license information see p.557

Bulk quantities and custom formulations available upon request

Table 1.2.FastDigest restriction enzymes.

(continued on next page)

(continued on next page)

For patent and license information see p.557

Bulk quantities and custom formulations available upon request

Table 1.2.FastDigest restriction enzymes.

Alphabetic list of commercially available restriction enzymes see p.207.

Single letter code

5...G pUC19 DNA/SmaI, 1.0% agarose 3...CT

A C G TC...3 G C A G...5 50l

Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml

1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.

Lambda DNA, 1g/20l 15 min

FastDigest AccI (XmiI)

TM K A C...3 A K MT G...5 #FD1484 50l

1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.

Lambda DNA, 1g/20l 5 min

T A C C...3 C A T GG...5 #FD0904 100l

1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.

Lambda DNA, 1g/20l 5 min

Protocols and Recommendations p.70

For patent and license information see p.557

Bulk quantities and custom formulations available upon request

FastDigest AciI (SsiI)

G C...3 G CG...5 #FD1794

1l of enzyme (1 FDU) cleaves 1g of lambda DNA in 5min at 37C in 1XFastDigest Buffer.

Supplied with: 10XFastDigest Buffer 1 ml 10XFastDigest Green Buffer 1 ml

Lambda DNA, 1g/20l 5 min

FastDigest AclI (Psp1406I)

1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.

Lambda DNA, 1g/20l 5 min

FastDigest AcuI (Eco57I)

Lambda DNA, 1g/20l 15 min

FastDigest AfeI (Eco47III)

1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.

Lambda DNA, 1g/20l 5 min

FastDigest AflII (BspTI)

T A A G...3 A A T T C...5 #FD0834 150l

1XFastDigest Buffer or 1XFastDigest Green Buffer at 37C.