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org/ at NATIONAL UNIVERSITY OF IRELAND, GALWAY on August 22, 2012 Published by Cold Spring Harbor Laboratory Press

Generation of Human scFv Antibody Libraries: PCR Amplification and Assembly of Light- and Heavy-Chain Coding Sequences
Jennifer Andris-Widhopf, Peter Steinberger, Roberta Fuller, Christoph Rader and Carlos F. Barbas III Cold Spring Harb Protoc 2011; doi: 10.1101/pdb.prot065573 Email Alerting Service Subject Categories
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cshprotocols.cshprotocols. Roberta Fuller. It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. the individual rearranged heavy.org MATERIALS RECIPES: Please see the end of this article for recipes indicated by <R>. Barbas III INTRODUCTION The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. Cold Spring Harbor Protocols RELATED INFORMATION Linkers of different compositions may result in different levels of oligomerization and may also alter the interface of the VH and VL regions. 1996.cshlp. 1996). Cite as: Cold Spring Harb Protoc.and Heavy-Chain Coding Sequences Jennifer Andris-Widhopf.). 2007).prot065573 © 2011 Cold Spring Harbor Laboratory Press www.and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning. 2012 Published by Cold Spring Harbor Laboratory Press Protocol Generation of Human scFv Antibody Libraries: PCR Amplification and Assembly of Light. A protocol describing the Generation of Human Fab Antibody Libraries: PCR Amplification and Assembly of Light. 2001.Downloaded from http://cshprotocols. 1993.org 1139 .and Heavy-Chain Coding Sequences (Andris-Widhopf et al. 1997).and heavy-chain variable regions are connected with a short peptide linker tend to form dimers.1101/pdb. coli by Electroporation (Sambrook and Russell 2006e). Alternative linkers that enhance scFv phage-binding activity have also been reported (Tang 1996. CSHL Press. Cold Spring Harbor. Christoph Rader. Zhu et al. USA. and Transformation of E. 2011. Barbas et al. In this method. but the use of a short linker can also lead to selection of unwanted low-affinity binders. whereas scFvs with long linkers tend to be monomers (Holliger et al. PROTOCOLS www.org/ at NATIONAL UNIVERSITY OF IRELAND. doi:10. The primers listed in this protocol are those used with the pComb3 vectors at the time of this writing. GALWAY on August 22. Bivalent diabodies can have the advantage of binding with higher avidity to their antigen. called bivalent diabodies. Agarose Gel Electrophoresis (Sambrook and Russell 2006b). 2011) is also available. Turner et al. McGuinness et al. Further information is also available on Quantitation of DNA and RNA (Barbas et al. Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c). and Carlos F. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). NY. Recovery of DNA from Agarose Gels: Electrophoresis onto DEAECellulose Membranes (Sambrook and Russell 2006d). Peter Steinberger. Adapted from Phage Display: A Laboratory Manual (ed. Single-chain fragments in which the light. One way to obtain these antibodies is through phagedisplay libraries constructed from human lymphocytes. as are protocols for Construction of cDNA Libraries Stage 1: Synthesis of First-Strand cDNA Catalyzed by Reverse Transcriptase (Sambrook and Russell 2006a).

org Table 1.. 2012 Published by Cold Spring Harbor Laboratory Press Reagents Agarose (Invitrogen) AmpliTaq DNA polymerase (Applied Biosystems) cDNA (see Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase [Sambrook and Russell 2006a]) DNA gel-loading dye (10×) <R> dNTP mix. XL1-Blue (Stratagene) Elutrap (Whatman) A QIAEX II Gel Extraction Kit (QIAGEN) can be used as an alternative in Step 3. e.ii. Vector. Applied Biosystems) www.cshprotocols. 5′ sense. 5′ sense.org/ at NATIONAL UNIVERSITY OF IRELAND. 10× (supplied with Taq polymerase) Restriction enzyme buffer M.Downloaded from http://cshprotocols. 100 mM. pComb3HSS or pComb3XSS (available from the Barbas laboratory) Equipment Electroporation apparatus and materials PCR cycler (e. Human scFv primers.5 mM (dATP/dCTP/dGTP/dTTP set. coli. long linker HSCVH1-FL 5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTG GTG CAG TCT GG 3′ HSCVH2-FL 5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG ATC ACC TTG AAG GAG TCT GG 3′ HSCVH35-FL 5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG GAG GTG CAG CTG GTG SAG TCT GG 3′ HSCVH3a-FL 5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG GAG GTG CAG CTG KTG GAG TCT G 3′ HSCVH4-FL 5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTG CAG GAG TCG GG 3′ (continued) www. 40 units/µL (Roche Applied Science) <R>SOC medium for phage libraries T4 DNA ligase.cshlp.. 5× (supplied with enzyme) TAE <R> Cold Spring Harbor Protocols Dilute the 50× stock to prepare 1× TAE electrophoresis running buffer.g. 2.org 1140 Cold Spring Harbor Protocols . short and long linker VH primers. electrocompetent. see Table 1) PCR buffer. 100-bp DNA (GE Healthcare) or 1-kb DNA (Invitrogen) Oligonucleotide primers (human Fab. GE Healthcare) E. LB agar + carbenicillin plates <R> MicroAmp PCR caps (Applied Biosystems) MicroAmp PCR trays (Applied Biosystems) MicroAmp PCR tubes (Applied Biosystems) Molecular weight marker. GALWAY on August 22. 1 unit/µL (Invitrogen) T4 DNA ligase buffer.g. short linker HSCVH1-F 5′ GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTG GTG CAG TCT GG 3′ HSCVH2-F 5′ GGT GGT TCC TCT AGA TCT TCC CAG ATC ACC TTG AAG GAG TCT GG 3′ HSCVH35-F 5′ GGT GGT TCC TCT AGA TCT TCC GAG GTG CAG CTG GTG SAG TCT GG 3′ HSCVH3a-F 5′ GGT GGT TCC TCT AGA TCT TCC GAG GTG CAG CTG KTG GAG TCT G 3′ HSCVH4-F 5′ GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTG CAG GAG TCG GG 3′ HSCVH4a-F 5′ GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTA CAG CAG TGG GG 3′ PROTOCOLS VH primers.cshprotocols. GeneAmp 9700. 10× (supplied with SfiI restriction enzyme) SfiI restriction enzyme.

i) 5′ CCT GGC CGG CCT GGC CAC TAG TGA CCT TGG GGC TGG TCG GGG ATG C 3′ HSCD-B (corresponding to CH1 domain of human IgD.cshprotocols. see Step 1.cshprotocols.org Vλ primers. 2012 Published by Cold Spring Harbor Laboratory Press Table 1.cshlp. see Step 1. GALWAY on August 22.i) 5′ CCT GGC CGG CCT GGC CAC TAG TAA GGG TTG GGG CGG ATG CAC TCC C 3′ HSCA-B (corresponding to CH1 domain of human IgA. 3′ reverse. short and long linker HSCJLam1236 5′ GGA AGA TCT AGA GGA ACC ACC GCC TAG GAC GGT CAS CTT GGT SCC 3′ HSCJLam4 5′ GGA AGA TCT AGA GGA ACC ACC GCC TAA AAT GAT CAG CTG GGT TCC 3′ HSCJLam57 5′ GGA AGA TCT AGA GGA ACC ACC GCC GAG GAC GGT CAG CTS GGT SCC 3′ Overlap extension primers RSC-F (sense) 5′ GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC 3′ RSC-B (reverse) 5′ GAG GAG GAG GAG GAG GAG CCT GGC CGG CCT GGC CAC TAG TG 3′ www.org/ at NATIONAL UNIVERSITY OF IRELAND. Continued HSCVH4a-FL 5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTA CAG CAG TGG GG 3′ VH primers.Downloaded from http://cshprotocols. 5′ sense.org 1141 Cold Spring Harbor Protocols . short and long linker HSCK1-F 5′ GGG CCC AGG CGG CCG AGC TCC AGA TGA CCC AGT CTC C 3′ HSCK24-F 5′ GGG CCC AGG CGG CCG AGC TCG TGA TGA CYC AGT CTC C 3′ HSCK3-F 5′ GGG CCC AGG CGG CCG AGC TCG TGW TGA CRC AGT CTC C 3′ HSCK5-F 5′ GGG CCC AGG CGG CCG AGC TCA CAC TCA CGC AGT CTC C 3′ Cold Spring Harbor Protocols Vκ primers. short and long linker HSCG1234-B (corresponding to human IgG isotypes 1-4) 5′ CCT GGC CGG CCT GGC CAC TAG TGA CCG ATG GGC CCT TGG TGG ARG C 3′ HSCM-B (corresponding to CH1 domain of human IgM. 3′ reverse. see Step 1. 3′ reverse.i) 5′ CCT GGC CGG CCT GGC CAC TAG TCA CAT CCG GAG CCT TGG TGG GTG C 3′ HSCE-B (corresponding to CH1 domain of human IgE.i) 5′ CCT GGC CGG CCT GGC CAC TAG TGA CGG ATG GGC TCT GTG TGG AGG C 3′ Vκ primers. 5′ sense. see Step 1. short and long linker HSCJK14o-B 5′ GGA AGA TCT AGA GGA ACC ACC TTT GAT YTC CAC CTT GGT CCC 3′ HSCJK2o-B 5′ GGA AGA TCT AGA GGA ACC ACC TTT GAT CTC CAG CTT GGT CCC 3′ HSCJK3o-B 5′ GGA AGA TCT AGA GGA ACC ACC TTT GAT ATC CAC TTT GGT CCC 3′ HSCJK5o-B 5′ GGA AGA TCT AGA GGA ACC ACC TTT AAT CTC CAG TCG TGT CCC 3′ www. short and long linker HSCLam1a 5′ GGG CCC AGG CGG CCG AGC TCG TGB TGA CGC AGC CGC CCT C 3′ HSCLam1b 5′ GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGC CAC CCT C 3′ HSCLam2 5′ GGG CCC AGG CGG CCG AGC TCG CCC TGA CTC AGC CTC CCT CCG T 3′ HSCLam3 5′ GGG CCC AGG CGG CCG AGC TCG AGC TGA CTC AGC CAC CCT CAG TGT C 3′ HSCLam4 5′ GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AAT CGC CCT C 3′ HSCLam6 5′ GGG CCC AGG CGG CCG AGC TCA TGC TGA CTC AGC CCC ACT C 3′ HSCLam78 5′ GGG CCC AGG CGG CCG AGC TCG TGG TGA CYC AGG AGC CMT C 3′ HSCLam9 5′ GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGC CAC CTT C 3′ HSCLam10 5′ GGG CCC AGG CGG CCG AGC TCG GGC AGA CTC AGC AGC TCT C 3′ PROTOCOLS Vλ primers.

Depending on the needs of the user.Downloaded from http://cshprotocols. www. 3–5).org/ at NATIONAL UNIVERSITY OF IRELAND. the purified VL and VH products are fused by overlap extension PCR using the sense and reverse extension primers. 1. and 27 Vλ amplifications (see Figs. The JL reverse and VH sense primers have overlapping sequence tails that code for the linker peptide in the final scFv PCR fragment. Cold Spring Harbor Protocols PROTOCOLS www. This protocol describes the construction of κ and λ human scFv libraries as separate entities. 16 Vκ amplifications. can be interrupted at any step. To amplify the V gene rearrangements for a scFv with a short or long linker. 2012 Published by Cold Spring Harbor Laboratory Press METHOD Figure 1 shows a schematic overview of the steps that are used to generate the PCR products. The VL sense primers include a 5′ sequence tail that contains an SfiI site and is recognized by the sense extension primer. the κ and λ light-chain products can be combined prior to the overlap extension PCR such that only one kind of reaction is needed. If good amplification is not obtained (if a strong amplified band is not clearly visible in the agarose gel). Generation of scFv fragments by PCR overlap extension for cloning into the pComb3 vector system. therefore. The resulting product is ~750–800 bp in size and is referred to as the scFv PCR fragment. The CH1 (or JH) reverse primers introduce a 3′ sequence tail that contains an SfiI site and is recognized by the reverse extension primer. perform 12 VH amplifica- tions. before or after SfiI restriction digestion.org 1142 Cold Spring Harbor Protocols . In the first round of PCR. The first-round products have a size of ~350–400 bp. GALWAY on August 22.cshprotocols. In the second-round PCR.cshprotocols. and the flowchart in Figure 2 depicts the entire library construction and selection procedure. lithium chloride precipitation of the RNA might improve the results (see Construction of cDNA Libraries Stage 1: Synthesis of First-Strand cDNA Catalyzed by Reverse Transcriptase [Sambrook and Russell 2006a]). It has asymmetric SfiI restriction sites on the 5′ and 3′ ends that are used for directional cloning into the pComb3 vectors.and heavy-chain variable regions are amplified by using VL and VH sense primers in conjunction with JL and CH1 reverse primers (or JH reverse primers in chicken scFv libraries). First Round PCR PCR products and other DNA samples. Alternatively. The construction of antibody fragment libraries. precipitated or in solution. A test amplification can be performed using the cDNA sample. the κ and λ scFv products can be combined at a later step. the rearranged light. can be stored for years at −20˚C.org FIGURE 1.cshlp.

Phage are rescued by the addition of helper phage and the panning/selection process is started using the rescued phage. this tail is recognized by the reverse extension primer used in the second-round PCR. Library construction begins with RNA preparation and cDNA synthesis. Each reverse primer has a sequence tail containing an SfiI site.org FIGURE 2.and IgM-specific primers is sufficient. PROTOCOLS FIGURE 3.cshlp.org/ at NATIONAL UNIVERSITY OF IRELAND. In most cases. 2012 Published by Cold Spring Harbor Laboratory Press Cold Spring Harbor Protocols www.cshprotocols. the use of the IgG. During this preparation process. www. GALWAY on August 22. The sense primers have a sequence tail that corresponds to the linker sequence used in the overlap extension PCR. pComb3 vector is also prepared and tested for ligation efficiency. Panning is a cyclic procedure that is carried out in consecutive rounds over the course of several days. Flowchart depicting library construction and selection. Each of the HSCVH-F (short linker) or HSCVH-FL (long linker) sense primers is paired with the reverse primers specific for the 5′ end of the CH1 regions of the different immunoglobulin isotypes. Prepared inserts are ligated into the prepared vector and transformed into competent bacterial cells.Downloaded from http://cshprotocols.cshprotocols. PCR amplification of human VH regions from cDNA. followed by preparation of PCR inserts for scFv (left) or Fab (right).org 1143 Cold Spring Harbor Protocols .

and long-linker libraries. The amplification of human Vκ sequences for the construction of scFv short.cshprotocols.org/ at NATIONAL UNIVERSITY OF IRELAND. using spleen/bone marrow cDNA as template: 1 µL 60 pmol 60 pmol 10 µL 8 µL 0.org 1144 Cold Spring Harbor Protocols .5 µg) 5′ primer (see primer combinations) 3′ primer 10× PCR buffer 2. GALWAY on August 22. short linker: HSCVH1-F HSCG1234-B www. this tail is recognized by the sense extension primer.Downloaded from http://cshprotocols.cshprotocols.org VH primer combinations. short and long linker: HSCLam1a HSCJLam1236 HSCLam2 HSCJLam1236 HSCLam4 HSCJLam1236 HSCLam78 HSCJLam1236 HSCLam1b HSCJLam1236 HSCLam3 HSCJLam1236 HSCLam6 HSCJLam1236 HSCLam9 HSCJLam1236 www.5 µL cDNA (0. VH primer combinations. 2012 Published by Cold Spring Harbor Laboratory Press FIGURE 4. Each of the HSCK-F sense primers is paired with each of the HSCJKo-B reverse primers to amplify human Vκ gene segments from cDNA. i. short and long linker: HSCK1-F HSCJK14o-B HSCK3-F HSCJK14o-B HSCK1-F HSCJK2o-B HSCK3-F HSCJK2o-B HSCK24-F HSCJK14o-B HSCK5-F HSCJK14o-B HSCK24-F HSCJK2o-B HSCK5-F HSCJK2o-B Vλ primer combinations. Assemble one reaction containing the following reagents for each primer combination. The reverse primers have a linker sequence tail that allows the overlap extension with the VH products in the second round of PCR.cshlp. long linker: HSCVH1-FL HSCG1234-B HSCVH35-FL HSCG1234-B HSCVH4-FL HSCG1234-B HSCVH1-FL HSCM-B HSCVH35-FL HSCM-B HSCVH4-FL HSCM-B HSCVH2-FL HSCG1234-B HSCVH3a-FL HSCG1234-B HSCVH4a-FL HSCG1234-B HSCVH2-FL HSCM-B HSCVH3a-FL HSCM-B HSCVH4a-FL HSCM-B HSCVH2-F HSCG1234-B HSCVH3a-F HSCG1234-B HSCVH4a-F HSCG1234-B HSCVH2-F HSCM-B HSCVH3a-F HSCM-B HSCVH4a-F HSCM-B HSCVH35-F HSCG1234-B HSCVH4-F HSCG1234-B HSCVH1-F HSCM-B HSCVH35-F HSCM-B HSCVH4-F HSCM-B PROTOCOLS Vκ primer combinations. The sense primers have a 5′ sequence tail containing an SfiI site.5 mM dNTPs Taq DNA polymerase Cold Spring Harbor Protocols Add H2O to a final volume of 100 µL.

We recommend using these reverse primers to build libraries for such specific purposes. as these are the isotypes that have the highest steady-state serum concentrations. which can generate nonspecific products. the VH sense primers listed below should be paired with the desired reverse primer as shown with IgM and IgG. GALWAY on August 22. ii.cshlp.org/ at NATIONAL UNIVERSITY OF IRELAND. Each of the HSCLam sense primers is paired with each of the HSCJLam reverse primers to amplify human Vλ gene segments from cDNA. this tail is recognized by the sense extension primer.and long-linker libraries.org 1145 Cold Spring Harbor Protocols . 72˚C for 90 sec 72˚C for 10 min A “hot start” PCR protocol can improve specificity.org Extra VH reverse primer sequences are provided in Table 1.cshprotocols. and yield. These primers correspond to the CH1 domain of human IgA. sensitivity. The three additional isotypes may be prevalent in certain types of responses. we make human libraries primarily from the IgM and IgG circulating pools of B lymphocytes. In each case. Traditionally. e. This protocol prevents low-stringency primer extension. These reverse primers can be used in the amplification of VH for both short linker and long linker scFv. The amplification of human Vλ sequences for the construction of scFv short. IgD. 56˚C for 15 sec.. www. The sense primers have a 5′ sequence tail containing an SfiI site. IgE antibodies are elevated in allergic responses and IgA is most prevalent at mucosal sites. HSCK1-F HSCJK3o-B HSCK3-F HSCJK3o-B HSCK1-F HSCJK5o-B HSCK3-F HSCJK5o-B HSCK24-F HSCJK3o-B HSCK5-F HSCJK3o-B HSCK24-F HSCJK5o-B HSCK5-F HSCJK5o-B HSCLam10 HSCJLam1236 HSCLam1a HSCJLam4 HSCLam2 HSCJLam4 HSCLam4 HSCJLam4 HSCLam78 HSCJLam4 HSCLam10 HSCJLam4 HSCLam1a HSCJLam57 HSCLam2 HSCJLam57 HSCLam4 HSCJLam57 HSCJLam78 HSCJLam57 HSCJLam10 HSCJLam57 HSCLam1b HSCJLam57 HSCLam3 HSCJLam57 HSCLam6 HSCJLam57 HSCJLam9 HSCJLam57 HSCLam1b HSCJLam4 HSCLam3 HSCJLam4 HSCLam6 HSCJLam4 HSCLam HSCJLam4 Cold Spring Harbor Protocols PROTOCOLS www. 2012 Published by Cold Spring Harbor Laboratory Press FIGURE 5. or a reversible inhibitor of the polymerase is used. The reverse primers have a linker sequence tail that allows the overlap extension with the VH products in the second round of PCR. In hot start PCR. and IgE.cshprotocols. either an essential reaction component is not added until the first denaturing step. Perform the PCR under the following conditions: 94˚C for 5 min 30 cycles of 94˚C for 15 sec.g.Downloaded from http://cshprotocols.

Run the products on a 2% agarose gel. Second Round PCR (Overlap Extension) In the second round of PCR. QIAEX II Gel Extraction Kit). expect a ~400-bp product for the VH reactions and a ~350-bp product for the Vκ and Vλ reactions. Equimolar quantities of light-chain variable and heavychain variable fragments are used to create the overlap extension product. 3. iii.. ii.5 mM dNTPs Taq DNA polymerase PROTOCOLS Add H2O to a final volume of 100 µL. Pool the products of each type of reaction. repeat the first round of PCR and combine the end products. 6).cshlp. Evaluate 5–10 µL of each reaction on a 2% agarose gel using DNA gel-loading dye and an appropriate molecular weight marker (MWM).org/ at NATIONAL UNIVERSITY OF IRELAND. cut out the correct-sized bands. and purify the DNA as described in Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-Cellulose Membranes (Sambrook and Russell 2006d). If yields are too low.5 µL 5′ primer (RSC-F) 3′ primer (RSC-B) 10× PCR buffer 2. unit = 50 µg/mL) (see Quantitation of DNA and RNA [Barbas et al. the appropriate first-round products are mixed in equal ratios to generate the overlap product. Isolate the PCR products: i. Perform overlap extension PCR: i. www. and wash as described in Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c). The primers in the first-round PCR create complementary sequences in the downstream region of the light-chain variable regions and the upstream region of the heavy-chain variable regions that serve as the overlap for the extension of the fulllength product (see Fig.g.D.D. 2012 Published by Cold Spring Harbor Laboratory Press 2. or resin binding (e. or by electroelution with an Elutrap. Approximately 2–4 µg of each pool is required to proceed.org Cold Spring Harbor Protocols 100 ng appropriate first-round products (see template combinations) 60 pmol 60 pmol 10 µL 8 µL 0.org 1146 Cold Spring Harbor Protocols . template combinations for scFv with a short linker: 100 ng short linker VH product 100 ng Vκ product 100 ng short linker VH product 100 ng Vλ product FIGURE 6. GALWAY on August 22. From these amplifications. Quantitate yields by reading the optical density (O. 2007]). ethanol-precipitate.) at 260 nm (1 O.cshprotocols. 4. The sense and reverse extension primers used in the second-round PCR recognize the sequence tails that were generated in the first-round PCR.cshprotocols. The second-round PCR to generate scFv PCR fragments.Downloaded from http://cshprotocols. Assemble ten 100-µL reactions for each overlap using the following reagents: www.

digest more PCR product with SfiI.: i. If yields are too low. (Optional) Ethanol-precipitate and wash as described in Standard Ethanol Precipitation of ˚ DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c). For each type of reaction. We recommend electroelution with an Elutrap (Whatman) for extraction of the vector from the gel. Isolate the digested products: i. Restriction Digestion 7. 8. the number of PCR cycles can be increased. Set up a digest of the vector containing the following reagents: 20 µg 120 units 20 µL 10× buffer M pComb3HSS or pComb3XSS (contains stuffer fragment between the two SfiI cloning sites) SfiI (6 units per µg of DNA) PROTOCOLS Add water to a volume of 200 µL. Isolate the PCR products as described in Step 3. expect a ~750. larger quantities of SfiI-cut and purified vector DNA can be prepared and tested in advance. The long PCR extension time favors full-length product. Incubate both digests for 5 h at 50 C. or more overlap PCRs can be performed. Prepare the overlap scFv PCR products and the pComb3HSS or pCOMB3XSS vector for cloning by Cold Spring Harbor Protocols performing restriction digests with SfiI. Suboptimal digestion or purification will yield vector with low transformation efficiencies and high background after ligation. ii. Perform the second-round PCR under the following conditions: 94˚C for 5 min 25 cycles of 94˚C for 15 sec.cshlp.i–3. iii. If the loss of digested PCR product in the final purification step is high or if several large-size ligations are needed to obtain a library of the desired size. www. If a strong band of the correct size is not clearly visible in the gel. Purify the digested scFv products on a 2% agarose gel. To retain diversity of the library. SfiI-digested DNA is partly insoluble after ethanol precipitation. 6. repeat the overlap PCR and combine the end products. Evaluate 5–10 µL of each reaction on a 2% agarose gel using DNA gel-loading dye and an appropriate MWM for reference. 2012 Published by Cold Spring Harbor Laboratory Press template combinations for scFv with a long linker: 100 ng long linker VH product 100 ng Vκ product 100 ng long linker VH product 100 ng Vλ product ii. and purify the vector and stuffer frag- ment on a 1% agarose gel (see Agarose Gel Electrophoresis [Sambrook and Russell 2006b]). GALWAY on August 22.org 1147 Cold Spring Harbor Protocols . The amount of added enzyme should not exceed 10% of the reaction volume. Set up a digest of the PCR products containing the following reagents: 10 µg 360 units 20 µL www. 72˚C for 2 min 72˚C for 10 min 5. it is better to increase the number of PCRs.cshprotocols. cut only once) and uncut vector DNA from the desired double cut product (size ~3400 bp).cshprotocols. Let the DNA run long enough to separate linearized vector DNA (size ~5000 bp. 10–15 µg should be sufficient to continue. In some cases. 56˚C for 15 sec.iii. ii.org/ at NATIONAL UNIVERSITY OF IRELAND. the DNA can be loaded directly after digestion onto the agarose gel (Step 8.ii).to 800-bp product. If desired. To avoid this problem.Downloaded from http://cshprotocols.org purified scFv product SfiI (36 units per µg of DNA) 10× buffer M Add water to a volume of 200 µL.

and plate 100 µL of each dilution on LB agar + carbenicillin plates. as follows: Small-scale test ligation (one reaction for each PCR insert): 140 ng 70 ng 4 µL 1 µL pComb3HSS or pComb3XSS. Ideally. if this number does not exceed 1 × 107. Dilute the transformed cultures 10-fold and 100-fold with prewarmed (37˚C) SOC. The vector preparation should contain little uncut vector DNA or DNA that is cut only once. SfiI-digested and purified scFv (short or long) overlap PCR product. the final library size should be several times 108 but at least 5 × 107 total transformants. SfiI-digested and purified 5× ligase buffer ligase ĆĆ Add H2O to a volume of 20 µL. Transform 0. 2007]).cshprotocols. 12. Incubate the plates overnight at 37 C. GALWAY on August 22. SfiI-digested and purified stuffer fragment. Test the ligation efficiency of the vector DNA by ligating it with the gel-purified stuffer fragment that is generated during the SfiI digestion of the vector DNA.Downloaded from http://cshprotocols.org 1148 Cold Spring Harbor Protocols . iii.org/ at NATIONAL UNIVERSITY OF IRELAND. Perform a small-scale test ligation in parallel with the two control ligations. as follows: Control ligation 2 (test for vector self-ligation): 140 ng 4 µL 1 µL pComb3HSS or pComb3XSS.cshlp. but there is not enough vector or insert to make a library of the desired size. iii. i. 11. Count the colonies on the vector + Fab insert plates and cal- culate the number of transformants per µg pf vector DNA. as follows: Control ligation 1 (control insert): 140 ng 140 ng 4 µL 1 µL pComb3HSS or pComb3XSS.i). The stuffer fragment is ~1600 bp. do not proceed with the large-scale library ligation. digested PCR products. Perform small-scale ligations to assess the suitability of the vector and inserts for high-efficiency lig- ation and transformation. SfiI-digested and purified 5× ligase buffer ligase PROTOCOLS Add H2O to a volume of 20 µL. If the background is >10% or the ligation efficiency is low.org Add H2O to a volume of 20 µL. If the ligations are reasonably good. Count the colonies obtained from Control ligation 1 and Control ligation 2 for an indication of vector ˚ quality and ligation efficiency. Estimate the amount of uncut or partly cut vector DNA by setting up a ligation reaction that Cold Spring Harbor Protocols contains only vector DNA. Quantify the purified. SfiI-digested and purified 5× ligase buffer ligase ĆĆ www. and include the same amount of vector DNA. vector. 2012 Published by Cold Spring Harbor Laboratory Press The stuffer fragment can be used in a test ligation to determine the quality of the vector prior to use in library ligations (Step 9. at 260 nm (see Quantitation of DNA and RNA [Barbas et al. perform additional digests.5–1 µL of each reaction by electroporation into 50 µL of XL1-Blue electrocompetent cells. iv. digest new vector and repeat the ligations. Incubate reactions between 4 h and overnight at room temperature. Determine the number of scaled-up library ligations needed to achieve the desired final library size. and stuffer fragment by measuring the O.cshprotocols. ii. www. Library Ligation 9.D. 10.

Hoogenboom HR. Russell DW. Snedecor B. ii.1101/ pdb. Holliger P. High level secretion of a humanized bispecific diabody from Escherichia coli. 2006a. Scott JR. McGuinness BT. SfiI-digested and purified scFv (short or long) overlap product. Russell DW. 16. Sambrook J.1101/pdb. www. Ethanol-precipitate and wash the pellet as described in Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c).9 mL glycerol 500 µL 10% (w/v) SDS 200 µL 0. Perform library ligation: 7 i.1101/pdb.org RECIPES Recipes for items marked with <R> are provided here. Sambrook J. Russell DW.cshprotocols. 2011.prot4456. Cold Spring Harb Protoc doi: 10. 2007. SfiI digested and purified 5× ligase buffer ligase Add H2O to a volume of 200 µL. J Biol Chem 271: 15682–15686.1101/pdb.4 µg 700 ng 40 µL 10 µL pComb3HSS or pComb3XSS. DNA gel-loading dye (10X) 3. FitzGerald K. 2006e. Quantitation of DNA and RNA. Transform the ligation reaction by resuspending in 15 µL of H2O and electroporating into XL1-Blue electrocompetent cells. Shuler P. 15. Cold Spring Harb Protoc doi: 10.org/ at NATIONAL UNIVERSITY OF IRELAND.prot3214. 2006b. Walter G. Bio/Technology 14: 192–196. George AJT. The ethanol-precipitated ligation reaction can be stored for months at −20˚C.025 g xylene cyanol Bring to 10 mL total volume with H2O. Incubate overnight at room temperature. “Diabodies”: Small bivalent and bispecific antibody fragments. 2006c. Proc Natl Acad Sci 90: 6444–6448. Jiang N. Duncan AR. Prospero T. 14. Standard ethanol precipitation of DNA in microcentrifuge tubes. Recovery of DNA from agarose gels: Electrophoresis onto DEAE-cellulose membranes. Proceed with the preparation and panning of the primary library. Cold Spring Harb Protoc doi: 10.025 g bromophenol blue 0. Mahoney W. Combine the following: 1. Barbas CF III. J Immunol Methods 205: 43–54.ip47. Cold Spring Harb Protoc doi: 10. Zapata G. Barbas CF III. Selection of linkers for a catalytic single-chain antibody using phage display technology.org/recipes. Russell DW. Rader C. 2012 Published by Cold Spring Harbor Laboratory Press A good vector DNA preparation should yield at least 108 colony-forming units (cfu) per µg of vector DNA and should have <10% (ideally <5%) background ligation (calculated as cfu per µg of vector DNA in Control ligation 2). Turner DJ. Shalaby R. as described in Transformation of E. Additional recipes can be found online at http:// www.org 1149 Cold Spring Harbor Protocols . 2006d. Parakh C.cshlp. 1996.1101/pdb. Agarose gel electrophoresis. Zhu Z. PROTOCOLS www. Sambrook J. 1996. Sambrook J. Carter P. Tang Y.prot4065.and heavy-chain coding sequences. Steinberger P. GALWAY on August 22. Nat Biotechnol 14: 1149–1154. Hilvert D. Silverman GJ. 13. Assemble enough reactions to produce at least 5 × 10 transformants.prot4020. Burton DR.cshprotocols. Cold Spring Harb Protoc doi: 10. Chen H. Russell DW. Phage diabody repertoires for selection of large numbers of bi specific antibody fragments.prot3933. Importance of the linker in expression of single-chain Fv antibody fragments: Optimisation of peptide sequence using phage display technology.5 M EDTA 0. Winter G. Cold Spring Harbor Protocols REFERENCES Andris-Widhopf J. Transformation of E.prot065565.1101/pdb. Construction of cDNA libraries stage 1: Synthesis of first-strand cDNA catalyzed by reverse transcriptase. Cold Spring Harb Protoc doi: 10. 1997.1101/pdb.Downloaded from http://cshprotocols. coli by electroporation. Ritter MA. Fuller R. coli by Electroporation (Sambrook and Russell 2006e). Sambrook J. Generation of human Fab antibody libraries: PCR amplification and assembly of light.cshprotocols. 1993. Cold Spring Harb Protoc doi: 10. 1996.

add 1 mL of carbenicillin stock solution (50 mg/mL). and titrate to pH 7.org 1150 Cold Spring Harbor Protocols . add the last two ingredients. Sterilize by autoclaving on liquid cycle for 20 min at 121˚C at 15 psi. stir until dissolved.5 M EDTA (pH 8. Stir and autoclave for 15 min at 121˚C.org/ at NATIONAL UNIVERSITY OF IRELAND. SOC medium for phage libraries 20 g tryptone 5 g yeast extract 0.5 g NaCl 186 mg KCl 10 mL MgCl2 (1 M.cshprotocols. sterile) 20 mL glucose (1 M.cshlp. When cooled to 45˚C-50˚C. GALWAY on August 22. Pour into Petri dishes and allow to solidify. combine 32 g of LB agar with 1 L of H2O.Downloaded from http://cshprotocols.0) The 1X working solution is 40 mM Tris-acetate/1 mM EDTA. 2012 Published by Cold Spring Harbor Laboratory Press LB agar+carbenicillin plates LB agar (GIBCO/Invitrogen) Carbenicillin (50 mg/mL) To prepare LB agar plates with 50 µg/mL carbenicillin. aliquot into 10-mL portions and store at room temperature. Store at 4˚C. Using aseptic conditions. filter-sterilized) Bring first 4 ingredients to 1 L total volume with H2O. When cooled. www.org PROTOCOLS www.cshprotocols.1 mL of acetic acid (glacial) 100 mL of 0. Cold Spring Harbor Protocols TAE Prepare a 50X stock solution in 1 L of H2O: 242 g of Tris base 57.