329 Plasmid and PCR Lab

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CHM329L Experiment #2

Plasmid Purification and Size Estimation
Objective: In this laboratory exercise you will purify a plasmid ifom E. coli using a alkaline lysis-based miniprep coupled with chromatography on a silica matrix, set up a restriction enzyme digestion, run an agarose gel, and be able to estimate the size of your unknown plasmid DNA that you just purified. Background: Plasmids are autonomously replicating circular DNA molecules found in many bacteria. They can be isolated from the chromosomal DNA by a variety of techniques. The number of plasmid molecules in a cell is determined by specific sequences on the plasmid, the origins of replication. High copy plasmids are found in large numbers in a cell and low copy plasmids are found in small numbers in the cell. Plasmids generally carry some type of gene that allows celIs to grow under conditions that normal cells without plasmids cannot. For example, many plasmids carry a gene conferring resistance of the cells to the antibiotic ampicllin. Thus, cells containing the plasmid can grow when arnpicllin is present while Wildtype cells would die. See the attached map of a common plasmid used in the lab, pUCI9. Plasmids are useful vectors or carriers for foreign DNA fragments. One can cut a plasmid with a restriction enzyme and insert a DNA fragment that was also cut with the same enzyme. This is the basis for cloning. When we construct plasmids during cloning, we oftenneed to estimate the size of the plasmid that we generate to confirm that a DNA fragment was inserted that was the correct size. A good way to do this is to cut the plasmid with a restriction enzyme that cuts the DNA only one time. This generates a linear piece of DNA that can be placed in an electrical gradient and separated according to size and charge. One can compare the migration of the plasmid DNA with DNA fragments oflcnown size and thus estimate the size ofthe plasmid. DNA molecules are generally separated using agarose gel electrophoresis. Agarose is a carbohydrate polymer that when heated in liquid and then cooled will form a gel-like solid. You can think of this like a mesh (Similar to polyacrylamide gels). DNA molecules which are negatively charged, will migrate M-omthe cathode to the anode when placed in an electrical field. Thus DNA molecules will travel through the agarose matrix at a rate that is based on their size and charge. Smaller molecules will travel faster than larger molecules. The DNA molecules can be visualized in the agarose gel by a variety of methods. One of the most common is to stain the DNA with an intercalating agent like ethidiurn bromide. Ethidium molecules will fluoresce brightly when exposed to UV light when intercalated between the bases of the DNA duplex, thus showing where the DNA fragments are in the geL The image of the fluorescent bands can be captured using photography or other image-capturing technology. You can estimate the size ofaDNA fragment by comparing its migration in the agarose gel with fragments oflrnown size (standards). A plot of the 10gMW of the DNA vs. Rm gives a standard curve from which you can determine the size of the unknown fragment
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Applications uSing QIAprep purified DNA Plasmid DNA prepared using the QIAprep syslem is suitable for a applications including:
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Resfriclionenzyme digestion library screening In vitro translation

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Sequencing Ligation and transformation Transfedion of robust cells

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Principle
The QIAprep miniprep procedure is based on alkaline lysis of bacterial cells followed by adsorption of DNA onlo silica in the presence of high soh (l J. The unique silica membrane used in QIApr!,!pMiniprep Kitscompletely replaces gloss or silica slurries for plasmid minipreps. The procedure consists of three bosic steps:
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Preparation and dearing of 0 boderiol lysnle

.,'" Adsorption of DNA onlo the QIAprep membrane ;,,, Washing and elution of plasmid DNA Alisieps are performed wilhout the use of phenol, chloroform, CeCl, elhldiurn bromide, and without alcohol precipitation.
Preparation and dearing of bac:teriallysote

The QIAprep miniprep procedure uses the modified alkaline lysis method of Birnbolm and Daly (2). Bacteria ore lysed under alkaline conditions, and the lysate is subsequently neutralized ond adjusted to high·salt binding conditions in one step. After lysate dearing, Ihe sample is ready for purification an the QIAprep silica membrane. Formore details on growth of bacterial cultures and alkaline lysis, please refer to Appendix A on pages 39-42. In the QIAprep Spin and alAprep 8 miniprep procedures, Iysotes are elected by centrifugation, while the QIAprep a and 96 Turbo Miniprep kits provide TurboFilter strips or plates for lysate dearing by filtration. f

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lyseBlue reogent· W r--, 't--Use of lyseBlue is optional and is not required 10 successfully perform plasmid prepa· otions. See "Using LyseBluereagent" on page 14 for more inFormalion.

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• Ly.eBlue reosent is only supplied with QIAprcp'Spin Miniprep Kit, since mulliwell or outomoled lormcts do n 01 ollow visucl control of individual samples.

QtAprep MiniprepHandbook

06/2005

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/LyseBlue is a color indicator which provides visual identification of optimum buffer mixing. This prevents common handling errors thai lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA, and cell debris -,This makes lyseBlue ideal for use by researchers who have not had much experience with plasmid prepcrotions as well as experienced scientists who wont to be assured ·of maximum product yield.

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DNA adsorption

to the QlAprep membrane

QIAprep columns, strips, and plctes' use a silica membrane for selective adsorption of plosrnid DI-.JA in high-salt buffer and elution in low-salt buffer. The optirnized buffers in the lysis procedure, combined with the unique silica membrane, ensure thai only DNA will be adsorbed, while RNA, cellular proteins, and metabolites are not retained on the membrane but ore found in the flow-through_

Washing and elution of plosmid DNA
Endonudeoses essential when derivatives, or Buffer PB wash culture volumes are efficiently removed by a brief wash slep with Buffer PB. This step is working with endN strains such as the JM series, HB 10 1 and its any wild-Iype strain, 10 ensure that plasmid DNA is not degraded. The step is also necessory when purifying low-copy plosmids, where large are used. by a brief wosh step wifh Buffer PE. High-gualily plasmid QIAprep column wilh 50-100 (.IIof Buffer EB or woler. The immediate use in a range of applications - no need to desoil.

Salts are efficiently removed DNA is then eluted from the purified DNA is ready for precipitale, concenlrate, or

Note: Elution· efficiency is dependent on ·pH_The maximum elution efficiency is achieved belween pH 7_0 and 8_5_ When using wafer for elution, make sure that the pH value is wilhin this ronqe. Store DNA at _20CC when eluted wllh wafer since DNA may degrade in lhe absence of a buffering agent. DNA yield Plasmid yield wilh the QIAprep miniprep system vorii1s depending on plasmid copy number per cell (see page 39), the individual insert in a plasmid, fodors that affed growth of the bacterial cullure [see pages 39-421, lha elution volume (Figure 1 I, and the elution incubation lime (Figure 2)_ A 1.5 ml overnight culture can yield from 5 to 151-'9 of plasmid DNA [Table 1, page 141_ To obtain the optimum combination of DNA quolily, yield,. and concentration, we recommend using Luria·Berloni (LB) medium for growth of cultures (for composition see page 4 T], eluting plasmid DNA in a volume of 50 pi, and performing a short incubation after addition of the elution buffer.

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60 . The graph shows Ihal on incubation time of 1 minute and doubling the elution buffer volume inereeses yield.<:: and Recovery _:-W 100 . 60 40 20 o 2 5 3 4 lncubcfion tim" [min) 10 30 • j Figura 2 ID fJ9 pBluescripl DNA wos pUrified using the QIAprep Spin Miniprep protocol and eluted olter Ih~ indicot.0 Cl '" g 0 u 0 " '" t: U 100 50 50 100 150 40 -< ~ 0 ~ 1 20 - ii! Elution v(Jlllm~ {fJl} Figure I 10 Vg pUC 1 8 D~IA was purified using the QlAprep Spin protocol and eluted with the indlcotcd volurnos of Bulfer Ell.Elution Volume versus DNA Ccncenlrofion QJAprep Spin '5: --.d incubofion lim es with eilher 50 fJl or 100 fJ1Buffer Ell. Incubation 100 EI Time versus DNA Recovery @: ~ > 11 ~ .£ 200 '.. 250 . Tha standard protocol uses 50 pi Buffer EB for elution. since this combines high yield wilh high ccncentmllcn. aD . However lhe yield can be increased by increosinglh" dution volume. QIAprep Miniprep Handbook 06/2005 13 .

.000 rpm (-17. mo eneous colorless s~s0 ension in~ as een effectively precipitated. ond DNA prepared using other methods. No. 4. bacterial ~ -#. the tube 4-6 times.. If the suspension can 0 . Lorge culture volumes (e.Pl and transfer a micro- Ensure that RNase A has been added to Buffer Pl.(Lw.. Mix gently by inverting the tube. A com pod while pellet will form.xed-tmtttal~. immediately after oddition of Buffer N3. Add 350 Vi Buffer N3 and mix immediately andilhoroughly by inverting. ?:5 mil may require inverling up' to 10 times. . Procedure 1. Centrifuge for 10 min ot 13.. Do not vortex. S pu 10 ~ (f7 ~ C cells in 250 pi Buffer. 3. mix the solution thoroughly.AtJ should be Visib~ '\ ) Ir-tyseBIIJ~een added to Buffer P T.pVl5 0.900 x gJ in a table-lop microcentrifuge. Add 250 VI Buffer P2 and mix thoroughly by inverting the tube 4-6 times. Do not qllow Ihe lysis reaction to proceed for more than 5 min.Protocol: Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrjfuge This protocol is designed for purificotion of up 10 20 jl9 of hiqh-copy plosmid DNA from 1-5 ml overnight cultures of E. To avoid localized precipitation. 22 QIAprep Miniprep Handbook 06/2005 .lhe suspension should be of blue hos gone on t e suspension IS c .. continue mixing the solution unti 0 0 ~~ IS achieved. ' .. Mixing should result iGneous y colored suspension.. reFer 10 lhe recommendations on poge 44. colorless regions or if brownish cell clumps ore \ ~till . ())DI'\Q_ 6--\ta. For purilicollon of low-copy plcsrnids and cosmids.' ~ IflyseBlue has been added to Buffer Pl the cell suspension wililurn blu ddi---" tion 0 P2.5.. e nclerio should be resuspended com lei 109 or pipetling up an cell dumps re 2. coli in lB [luric-Benoni] medium. Please read "Important Notes" on pages 15-21 before starting. IV' . large plasm ids (> 10 kb]. continue inverting the lube unlil the solution becomes viscous and slightly deor.d. The solution should became cloudy. Resuspend··pen~ted centrifuge tube. Note: All protocol sleps should be carried out at room lemperature. as this will result in shearing of genomic DNA If necessary.4.g. ~ ~ ~ If}$Nc.c~!umps after resuspension of the pellet. vigorously shake I llfferb'ot· tie 10 ensure LyseBlue p~mplat . If reagent has been used.j I~ .

. Plasmid DNA Purification Using the QZ' pre Spin Minipr °t and 5 ml Collection Tubes The QIAprep Spin Minipre rocedure can be performed using 5JP cenlrifuge tubes [e. " Steps 7 For woshins. Host slroins ~--1-BfiJ8 and DH5a'U do not require lhis odciilionol wos step. S. 6. Discord lhe llow-throuqh. Centrifug"e for 30-60 from step 4 fa the QIAprep spin column by decanting or s. HB101 oniits-tl all any wild-Iype strain.:.) at 3000 x 9 far 1 mr Step 9: Tr s ar the QIAprep spin column 10 a microcentrifuge lube.} . c~t. Pro1oca . To elute DNA..5. odd §O-p1'Buffer EB (10 mM Tris·CI. spin column by adding 0..lgh. nd 7~~ed: Wash !he QIAprep spin column by adding 0. should be fa 6wed with the follOWing modifications: Step 4: Place a QIAprep coJ/ection lube.q. Place the Q]Aprep column in a cleon 1. The standard protocol on pages 22. QIAprep Miniprep Hondbcck 06/2005 23 . Discord the now·thraugh. 115101 0 15261) as collection lubeylo decrease hondlinq.-sf ps. to. Greiner.5 ml Buffer P centrifugingtm-s 5. Apply the supernatanis pipelfing. 0 f'\\-\ I This step is necessary to remove dease ocr '1yW en using endN slroins such as the JM series. Important: Residual wash buffer will not he completely removed unless the now-Ihrough is discarded before this additional centrifugation_ Residual alhcnol from Buffer PE may inhibit subsequent enzymatic reocllons. ret stond for 1 min. Wash QIAprep 30-60 5. and centrifuge for 1 min. Beckmon" Step 6: Centrifuge at 3000 x 9 ~ GS-6KR centrifuge at 00 rpm). Centrifuge of aximum speed for 1 min. Continue wilh slap lOaf Ihe protocol.75 ml Buffer PE and centrifuging for 9. spin column. and centrifuge for an additional "1 min fo remove residual wash buffer. centrifugalion should be performed and B: (The flo through does not need 10 be discarded. [The Row-through does nor-~ed to be discorded. pH 8._g.5 ml microcentrifuge lube. Discard Ihe flow~lhrol.. spin column in a i min using a suitable rotor (e.51 or wafer to Ihe center of each QIAprep /00 vl. no. which hove high levels _9Elet:1SeOdlvify or high carhohy ro nlenl.

5 mg/mL ethidium bromide. Be very careful as agarose solutions become easily superheated and will explode out of the bottle when swirled. Place the uncut tube on ice. by your lab instructor. 3.\.t. To the "EcoRI" tube. ideally to 55°C. Restriction Enzyme Digest I. Pour the contents slowly into the prepared casting stand.~ C. swirl contents of bottle. To each tube add the following: 14. Cap the tube tightly and place it in the 37°C water bath for 1 hour.5X E buffer into a 200 mL flask or 200 mL battle if available. Fill the gel box with O. Place the gel into the gel box so that the well end of the gel is nearest the black electrode. It is now ready to load with samples. \aL . and continue microwaving in 15 second intervals until all agarose is melted. of the EcoRI enzyme. Set up the gel casting stand as described 2. You will pour one minigel for each two groups in the lab. Place 50 mL ofO. Then add 2. Let the mixture gel for about 15-30 minutes (it will take on a turbid appearance when cool).5X E buffer so that the gel is completely submerged in buffer. briefly spin down the tube in the microcentrifuge to bring all the droplets to the 1 bottom of the tube. Remove any bubbles with a Pasteur pipet. Remove the comb from the gel by holding it at each end and wiggling it back and forth gently.0 !JL ofunlcnown plasmid DNA (-0. Obtain 2 clean sterile microfuge tubes for each plasmid sample you are analyzing. handled. Unclamp the gel from the casting stand.~ Cs~tlJ2. Preparation of1% (w/v) agarose gel with 0. Microwave on high for 45 seconds._' CHM329L Experiment #2 Dr. Take care not to rip it violently from the gel or you may cause the gel wells to tear.0 ul.0 /-lL of buffer/water mixture 5. Label bath tubes with unknown code and one tube as "uncut" or "Ecolcl".5 g of agarose powder to the buffer. ? . If your tube has liquid droplets on the sides after you place the reagents in it.FrH. Add 0. Let the solution cool until the bottle can be easily .5 ug) 2.5 ilL ofEthidium bromide (25 mglmL) to the solution and swirl gently to distribute the dye (Gloves should be worn as Ethidium bromide is mutagenic).ro~{) o-i-~ S. Alex B. have the instructor add 1. Pouring the gel: I.

Calculate the Rm for your EcoRl-cut DNA fragments (not the uncut ones). Keep track of which wells have which samples . To both of your tubes. From the standard curve. 1. start the current by hitting the front button on the power supply (has a picture of a running man). into the end well (instructor will heat samples). Calculate the Rm for each ofthe standard DNA }lands that are clearly visible on the gel. Plot the log (size of the DNA standard in basepairs) vs .1 of lOX Loading Dye. What is the purpose of adding NaOH/SDS to the resuspended bacterial pellets in your purification procedure? Why is is necessary to control the amount of time that the extract is exposed to this reagent? . you should see that the uncut band migrates at what appears to be a faster rate than the linear fragment Can you think of any reason why the uncut circular DNA isolated from the bacterium appears to be smaller than the cut DNA? .. E. uncut in one lane and cut in another lane. 3. estimate the size of your DNA fragments in basepairs.' CHM329L Experiment #2 Dr. and run the gel until the loading dye migrates 112 to 3/4 of the way down the gel (1-1. 2. Analysis of Results: This is for your report .4. Place the lid on the box and tum on the power supply (switch on right side). 5. Tum off the power supply and disconnect it from the gel box. 5. Dump the used gel into the waste container. Indicate the unknown code for the estimated size of your plasmid... Alex D. rinse the gel box and tray with distilled water. 4. You may not get a perfectly straight line. Running the gel 1. add 3 )1. Load your samples in adjacent lanes. . Rm is the ratio ofthe distance traveled by the DNA from the wells ldistance traveled from wells to thedye front.5 hours). Set the voltage for 80V (using arrow keys). Remove your EcoRl digest from the water bath. ·6. wear gloves when handling the gel and buffer. Please write an outline of the procedure you used to isolate your plasmid DNA. Heat the DNA standards for 2-5 minutes at 65°C and then load 20 ul.Rrn. Looking at the gel. Remove the gel tray from the box and have your instructor help you photograph it. 2. 3.

686 4. Fragment 1 2 Base Pairs 8.264 702 224 117 3 4 5 6 7· 8 9 10 II 12 13 14 4 . In general.371 1. Alex Size of Lambda BstEII cut DNA (Standards) In a typical 1% agarose gel.929 1.242 6. you will be able to see the 702 up to 3. and those bands should be used to construct the standard curve.· r Cl-JM329L Experiment #2 Dr.324 3.822 4. .454 7.323 1.675 2.369 5.675 bands clearly on these gels. you should see the smallest 12 of the 14 fragments generated.

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Centrifuge for 30-60 5_ (from step 4J to the QIAprep spin column by decanting or Discord the f1ow·through. transfer to a Procedure 1.IIBuffer EB or waler to the center of each Q1Aprep spin column.000 rpm (-17.Ig high·copy plasmid DNA from 1-5 ml overnight E. Centrifuge for 10 min at 13. Check BuFfers P2 and N3 for solt precipitation 3rc iFnecessary. and centrifuge for 1 min. / . If using lyseBlue reagenl. co/. by inverting the tube 4-6 4. Wosh QIAprep spin column by adding 30-605.Bench Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge QIAGEN ••••• ••••• This protocol is designed for the purification of up to 20 ). Resuspend pelle led bacteriol microcentrifuge tube. Optional: Add LyseBlue reagent to Buffer Pl. ond redissolve at Add ethanol (96-100%) to Buffer PE. 2. cells in 250 ).5 ml microcentrifuge tube. 2nd ed. 9. 7. ~ . solution turns colorless.II Buffer PI and Add 250 III Buffer P2 and mix thoroughly by inverting the tube 4-6 times. let stand for 1 min. culture in LB medium. and centrifuge for an additionall wash buffer. If using LyseBlue reagent. New users and users wonting to purify low-copy plasmids and cosrnids. This step is only required when using endA< or other bacteria strains wilh high nuclease activity or carbohydrate contenl (see QIAprep Miniprep Hondbook for more details) B. min 10 remove residual 10. Add 350 III Buffer N3 and mix immediately and thoroughly times. 3. Discord the flow-through. large plcsmids (:> 10 kb]. and DNA prepared using ather methods should refer to lhe detailed protocols provided in the QIAprep Miniprep Handbook.5 ml Buffer PB and centrifuging for 30-60 s. 5. ploce the QIAprep column in a dean 1. Recommended: Wash the QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging For Discard the f1ow·through. To elule DNA. 6. Add 50 ). solution turns blue. Things 10 do before starting • II • B Add RNase A solution to Buffer Pl. 0.900 x gJ in a loble·lop microcentrifuge. Apply the supernatant pipelting.

then what controls these differences and whatis the blueprint for this difference? That answer lies within our genome.understanding the mechanisms that control how DNA is translated into proteins within living cells. the complete answer to how and why does not lie solely in the knowledge of how enzymes runction. from generation to generation.-Student Manual Introduction to PCR The Polymerase Chain Reaction You are about to perform a procedure known as PCR1 to amplify a specific sequence of your own DNA in a test tube.secrets held in this most basic building block of life? Before Beginning This Lab. propagate. scientists have spent many lifetimes studying' proteins in an attempt to understand how they work. A researcher can use tiny amounts of genomic DNA 39 . prevent. transformed molecular biology into a multidisciplinary research field within 5 years of its invention. Molecular biology is the study of genes and the molecular details that regulate the flow of genetic information from DNA to RNA to proteins. In the last 20 years. and tendencies. The objective of PCR is to produce a large amount of DNA in a test-tube (in vitro). of interest. Biotechnology uses this knowledge to manipulate organisms' (microbes. called the polymerase chain reaction (peR). such as genomic DNA. people. or genetic code. Technically speaking. However. and overcome disease and physical handicaps as well as explain exactly how and why organisms exist. it was believed we could cure. I . If each enzyme is. and proteins are closely tied to each other. DNA. Thus. and die.dlttersnt. Before peR. or animals) DNA 10 help solve human problems. With this understanding. study nucleic acids as well as proteins to get a complete picture. See If You Can Answer the Following Questions . Kary Mullis2 at Cetus Corporation developed the molecular biology technique that has since revolutionized genetic research. you may realize why researchers today. in an attempt to understand the mechanisms behind the various biological processes. but not all. The genome. starting from only a trace amount. this means the controlled enzymatic amplification of a DNA sequence. RNA. Within the molecular framework of biology. composed of DNA.. many advances in nucleic acid techniques have allowed researchers to study the roles that nucleic acids play in biology. is our hereditary code. we must learn how they are made. Because proteins and enzymes ultimately play such a critical role in the life process. the molecular biology techniques used to study DNA required such a high level of expertise that relatively few scientists could use them. behavior. plants. PCR Set the Stage for a Scientific Revolution In 1983.~ How is DNA faithfully passed on from generation to generation? What causes genetic disease in some people but not others? How do scientists obtain DNA to study? What secrets can DNA tell us about our origins? What human problems can an understanding of DNA help us solve? Should we unlock the. It took the imagination and hard work of many scientists to reveal the answers to one of the most mysterious puzzles of life . You will be looking for a particular piece of DNA that is present in the genes-of many. This technique. or gene. This is the so-called blueprint that controls much of our appearance. Analysis of the data generated in this laboratory will enable you to determine whether or not you carry this specific DNA sequence. The template strands can be any form of DNA.

PCR is now used as a medical diagnostic tool to detect specific mutations that may cause genetic disease:" in criminal investigations and courts of law to identify suspects. Two methods for DNA template preparation are provided in the manual. pharmaceutical. and make enough DNA to study. Your instructor will indicate which exercise to follow. 40 . The development of peR transformed molecular biology from a difficult science to one of the most accessible and widely used disciplines of biotechnology. agricultural. DNA sequencing. forensic. only a single template strand is needed to copy and generate millions of new identical DNA molecules." and in the sequencing of the human qanorne. the use of molecular biology techniques for therapeutic. PCR has made an impact on four main areas of genetic research: gene mapping. let's extract some of your awn DNA. Prior to PC8.from a drop of blood. or medical diagnostic purposes was neither practical nor cost-effective. Now. or a cheek cell. and gene detection. In theory. It is the ability to amplify the precise sequence of-DNA of interest that is the true power of PCR." Prior to PCR. cloning. this would have been impossible. a single hair follicle.

Your cheek cells will then be lysed. When you rupture the cells. by heating to release all of their cellular constituents. which are used by cells to digest the DNA of invading viruses. After this 10 minute incubation period. or ruptured. such as Mg2+.Lesson 1 Cheek Cell DNA Template Preparation To obtain DNA for use in the polymerase chain reaction (PCR) you will extract the DNA from your own living cells. you will use the following procedure: The cheek cells are transferred to a micro test tube containing InstaGene™matrix. You will first suspend your isolated cheek cells in the InstaGene matrix and incubate them at 56°C for 10 minutes. In this lab activity. metal ions out of solution. Y{)U will use the extracted genomic DNA as the target template for PCR amplification. when the cells are lysed in the presence of the chelating beads. Boiling ruptures the cells and releases DNA from their nuclei. To obtain pure DNA for PCR. these DNases can digest the released DNA. or grab. such as DNases. This preincubation step helps to soften plasma membranes and release clumps of cells from each other. It is interesting to note that DNA can be also extracted from mummies and fossilized dinosaur bones. you will rinse your mouth with a salinetsalt) solution. including enzymes that were once contained in the cheek-celllysosames. Lysosomes are sacs in the cytoplasm that contain powerful enzymes. and collect the cells using a centrifuge. The heat also inactivates enzymes. However. which are required as catalysts or cofactors in enzymatic reactions. You will then boil the cells to rupture them and release the DNA they contain. It traps metal ions. To do this. This virtually blocks enzymatic degradation of the extracted DNA so you can use it as the template in your PCR reaction. place the cells in a boiling (100DG) water bath for 5 minutes. -' 41 . such as DNases. the cofactors are adsorbed and are not available to the enzymes. you will isolate DNA from epithelial cells that line the inside of your cheek. This particulate matrix is made up of negatively charged. microscopic beads that chelate. which can degrade the DNA template.

one that is complementary to a portion of one 'strand of the template. 1. the reaction mixture requires the following components: 1. DNA strand synthesis The two DNA primers provided in this klt are designed to flanlt a DNA sequence within your genome and thus provide the exact start signal for the DNA polymerase to "zero in on" and begin synthesizing (replicating) copies of that target DNA. Magnesium ions . In this way. Individual deoxynuc!eotides . When all the other components are combined under the right conditions. The complete master mix contains Taq DNA polymerase._ raw material of DNA 3. Taq DNA polymerase extends the annealed primers by "readinq" the template strand and syntheslzlnq the complementary sequence. To replicate a piece of DNA.576 exact copies of the target sequence. copies are made from copies and the number of template strands doublesfrom 2 to 4 to B to 16 and so on . magnesium ions. deoxynucleotides. DNA polymerase . This polymerase was isolated from a heat-stable bacterium (1hermus aquaticus) which in nature lives within high temperature steam vents such as those found in Yellowstone National Park. In this activity. you will amplify a region within your chromosome 16. T. Complementary strand hybridization takes place when the two different primers anneal.048.OOG-50. The primers are two short Single-stranded DNA molecules (23 bases long).OOO individual genes in the human genome. 50 . Salt buffer provides the optimum ionic environment and pH for the PCR reaction The template DNA in this exercise is genomic DNA that was extracted from your cells.pieces of DNA complementary to the template that tell DNA polymerase exactly where to start making copies 6. and buffer. G. Oligonucleotide primers .Lesson 2 peR Amplification It is estimated that there are 30. and C) . and another that is complementary to a portion of the opposite strand.doubling the number of template strands. The recipe for a PCR amplification of DNA contains a simple mixture of ingredients.an enzyme that assembles the nucleotides into a new DNA chain 4. Each time this cycle is repeated. or bind to each of their respective complementary base sequences on the template DNA. Complementary DNA strand hybridization via DNA polymerase that cells use to duplicate their 2.until after 20 cycles there are 1.(A. a copy of the original double-stranded template DNA molecule is made .containing the intact sequence of DNA to be amplified 2. Taq polymerase replicates the two template DNA strands. oligonucleotide primers. These primers anneal to the separated template strands and serve as starting points for DNA Taq replication by DNA polymerase.5 For this reason these enzymes have evolved to withstand high temperatures (94°C) and can be used in the PCR reaction. DNA template . ' PCR makes use of the same basic processes DNA. The true power of PCR is the ability to target and make millions of copies of (or amplify) a specific piece of DNA (or gene) out of a complete genome.a cofactor (catalyst) required by DNA polymerase to create the DNA chain 5.

annealing. and extension form one "cycle" of PCR. TemperatureCycle - = Denaturation Step (94"C)+ Annealing SIEp (BOaC) + Extension Step (naG) 52 . The entire 40 cycle reaction is carried out in a test tube that has been placed into a thermal cycler. the temperature that produces optimal Taq polymerase activity. A complete PCR amplification undergoes 40 cycles. The three steps of denaturation. The rapid heating and cooling of this thermal block is known as temperature cycling or thermal cycling.polymerization the reaction temperature is 72°C. The thermal cycler contains an aluminum block that holds the samples and can be rapidly heated and cooled across broad temperature differences.

The larger chromosomes contain more DNA. these differences in our DNA represent the molecular basis of DNA fingerprinting used in human identification and studies in population genetics. but within genes. it contains both coding and noncoding sequences. 10. splitting them into segments. while exons do nat.000-50. However. When RNA is first transcribed from DNA.~ . compared to the smaller chromosomes. = Intron 1 Inlron 2 3' Genomic DNA 5' t 5' Exon 1 Exon2 Trnnscnptlon 3' Pm-mRNA Genotypa t t Splfcing I ExonJ mRNA Translation Protein sPhenotype Fig.000 genes only comprise 5% of the total chromosomal DNA. The exact function of the noncoding DNA is not known.Lesson 3 Gel Electrophoresis of Amplified peR Samples and Staining of Agarose Gels What Are You Looking At? Before you analyze your peR explored. of the human genome (23 chromosomes come from the mother and the other 23 come from the father) contain approximately 30. let's take a 1001< at the target sequence being What Can Genes and DNA Tell Us? It is estimated that lhe 23 pairs. lntrons often vary in their size and sequence among lndividuals. Splicing of introns from genes. products.000 genes. the noncodong introns (in stay within the nucleus) are removed from the RNA while the exons (ex = exit the nucleus) are spliced together to farm the complete messenger RNA coding sequence for the protein (Figure 10). the 30. These mutations in our noncoding DNA are silently passed on to our descendants. 57 . This non coding DNA is found not only between.000-50. although it is thought that non coding DNA allows for the accumulation of mutations and variations in genomes. Each of the homologous chromosome pairs contains similar genes. we do not notice them because they do nat affect our phenotypes. Each gene holds the code for a particular protein. and therefore more genes. While the RNA is still in the nucleus. Interestingly. This process is called RNA splicing and is carried out by specialized enzymes called spliceosomes. The other 95% is noncoding DNA. or 46 chromosomes. Each chromosome contains a series of specific genes. This variation is thought to be the result of the differential accumulation of mutations in DNA throughout evolution.

Since you are amplifying a region of DNA contained within an intron. The gel will reveal two bands for such a sample. analysis of a single Alu repeat is used to estimate its frequency in the population and as a simple measure of molecular genetic variation . In this exercise. or 941 base pairs long if Alu is present. In this laboratory activity you will look at an Alu element in the PV92 region of chromosome 16. On a gel they will migrate at the same speed so there will be one band that corresponds to 941 base pairs. The presence or absence of this insert can be detected using peR followed by agarose gel electrophoresis. while some may not have the insert on either copy of the chromosome (Figure 12). If neither chromosome contains the insert. each amplified peR product will be 641 base pairs and they will migrate as one band that corresponds to 641. So if you don't have it. Some of these Alu sequences have characteristics that make them very useful to geneticists.P The origin and function of these repeated sequences is not yet known. . If both chromosomes contain Alu inserts. This particular Alu element is dimorphic. s· 3' In Iron Fig. 58 . three distinct outcomes are possible. 11. don't worry. Some people have the insert in one copy of chromosome 16 (one allele).with no reference to disease or relatedness among individuals.The Target Sequence The human genome contains small. each amplified peR product will be 941 base pairs long. base pairs. . they can either be associated with a disease or be used to estimate relatedness among individuals. This increase in size is due to the 300 base pair sequence contributed by the Alu insert. When your peR products are electrophoresed on an agarose gel. meaning that the element is present in some individuals and not others.000 times throughout the human qenome. others may have the insert in both copies of chromosome 16 (two alleles). there will be one peR product of 641 base pairs and one of 941 base pairs. If there is an Alu insert on one chromosome but not the other. This is a DNA sequence about 300 base pairs long that is repeated almost 500. . the region of DNA is never really used in your body. When present within introns of certain genes. One such repetitive element is called the "Alu sequence'? (Figure 11). Location of an Alu repetitive element within an intron. The primers in this kit are designed to bracket a sequence within the PV92 region that is 641 base pairs long if the intron does not contain the Alu insertion. repetitive DNA elements or sequences that have became randomly inserted into it over millions of years.

5. Fragments of the same size stay together and migrate in what appears as a single "band" of DNA in the gel. Over a period of time. 12. J ~ Hornozyqous t+r+] 941 base pairs .. which is placed into a chamber filled with a conductive buffer solution.1 " j F ! Ci-. The presence or absence of the Alu insert within the PV92 region of chromosome 16.' II.i. I Homozygous [-1-) 641 base pairs Heterozygous [+1-) 941 and 641 base palrs ~ " Fig. which contains 1. and 100 bp fragments (lane 1). two homozygous (+/+) individuals with 941 bp fragments (lanes 2. DNA fragments are loaded into an agarose gel slab.6). and two heterozygous (+/-) individuals with 941 and 641 bp fragments (lanes 4 and 7). 1 2 3 4 5 6 7 8 (bp) . three homozygous (-1-) individuals with 641 bp fragments (lanes 3.000 I" . Electrophoretic separation of DNA bands based on size. and B). In the sample gel below (Figure 13). 700 bp. j -. 59 ~( . 200 bp.000 bp. . and when placed in an electric field will be drawn toward the positive pole and repelled by the negative pole. Electrophoresis separates DNA fragments according to their relative sizes. EZ Load DNA molecular mass ruler. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. 1. :1 700 500 200 100 Fig. A direct current is passed between wire electlodes at each end of the chamber. 13. 500 bp. DNA fragments are negatively charged.II II· II :1 G~nDtyp" DNA Si>:~ of peR Produ<ls "I' i! . smaller fragments will travel farther than larger ones. peR-amplified bands of 941 bp and 641 bp are separated based on their sizes.

that can be selected and used for fingerprinting analysis. By comparing your DNA migration pattern to the controls. there are hundreds of loci. one needs to ask the question: Statistically. There are three possible genotypes for the Alu insert at the location you have amplified. some populations show much less variation in particular DNA segments than others. like Alu. then little information will be obtained from using that segment alone to identifY an individuaL In such a case. If your sample lane is blank. For DNA fingerprinting to be admissible as evidence in court. it must analyze 30 to 40 different DNA segments on multiple chromosomes from the same person.B The data from these studies have been published. Analysis Compare your sample lanes with the control lanes using the DNA size marker as a reference. A lower degree of variation will increase the odds of more than one individual having the same sequence. determine the number of individuals of each genotype: homozygous +/+. or DNA segments. homozygous -/-. or heterozygous +l-.OOO. discuss the possible reasons with your classmates and teacher. The Alu insert you have fingerprinted in this exercise has been used to study the migration patterns of human populations over time. and heterozygous +/-.000? 1 in 10? 1 For a DNA fingerprint to identify a suspect in a criminal case or a father in a patemity suit. being studied.Lesson 4 Analysis and Interpretation of Results If the overnight staining protocol was used to slain the gels. but closer to a 1 in 10 million (1/107) chance of a match. Tally the class results in the table on page 70. determine whether you are homozygous +/+. Depending on demographic factors such as ethnicity and geographic isolation. 69 . If 33% (1 out of three individuals) of a given population has the same fingerprinting pattern for a certain DNA segment. a positive result would only identify a person with 33% accuracy. The frequency of a particular DNA pattem turning up in a population drastically decreases when multiple DNA segments are selected and amplified. rather than just one segment. accurate identification required not a 1 aut of 3 (1/3) chance of a match in a population. Mark the location and size of your fragment or fragments. For a class. homozygous -/-. In humans. A major factor affecting the reliability of DNA fingerprinting evidence in forensics is population genetics and genetic statistics. Remember that the interpretation of this gel allows you to determine your genetic makeup only at the locus (chromosomallocalion). and your class samples can be compared to the data collected' from much larger populations. how many people in a papulation have the same DNA pattern as that taken from a crime scene: 1 in 1. record your results and dry your gels as described earlier. In analyzing how incriminating the DNA evidence is.OOO? in 10.

The frequencies of genotypes and alleles are basic characteristics that population geneticists use to describe and analyze populations. +1-. What are the genotypic frequencies of +1+. -1-) may exist in equilibrium. Table 1. the individuals may all be homozygous -1-. In a "meltinq-pot" population. and -/-. It is simply a sequence that can be used to study human genotypic frequencies. you can count up the alleles of your class "population" and determine their frequencies.Lesson 4 Analysis and Interpretation of Results Focus Questions Remember that this Alu sequence is inserted into a non coding reqion of the PV92 locus on chromosome 16 and is not related to a particular disease. +1-. the three genotypes (+1+. in some small." 1. I. geographically isolated populations. all individuals may be homozygous +1+. You can then compare the allelic and genotypic frequencies of your class population to published reports of larger population sizes. I' I Total = = 1. and -/.00 70 .in your class population? Fill in the table below with your class data. In others. nor does it code for any protein sequence. t [I The results of the peR reactions reveal your and your classmates' genotypes: +/+. The results you obtain in this exercise provide a real-life opportunity to calculate genotypic and allelic frequencies of the Alu insert in your class and to use the Hardy-Weinberg equation. +/-. the Alu insert in chromosome 16 is very useful for the study of gene frequencies in localized human populations: Theoretically. Knowing your genotypes. Because Alu repeats appear in the general population at random. Observed Genotypic i Frequencies for the Class Frequency (# of Genotypes/TotaJ) Category Homozygous (+/+) Heterozygous (+/-) Homozygous (-/-) Number Ii ! !. What is your genotype for the AIU insert in your PV92 region? I j I 2.

Table 3. Allele frequencies can be calculated from either the numbers or the frequencies of the genotypes (since they are related to each other)..students) total number of alleles (both + and-) frequency of (+/+) students + % (frequency of (+/-) students) number of H alleles total number of afJeles (both + and -) 2 (# of -/. respectively.422 5.students) + 1 (# of +/.------= 1.24 0. What is the frequency of each allele in your class sample? Fill in the table below with your class data. Calculated Allelic Frequencies Category (+) alleles (-) alleles Total alleles Number for the Class Frequency p q =: = --!.students) total number of alleles (both + and -) frequency of (-/-) students + % (frequency of (+/-) students) = q = frequency of (-) allele = = =: 3.000 0. The following table presents data from a USA-wide random population study.Allelic frequencies can be calculated from the numbers and frequencies of the genotypes in the population. p = frequency of (+) allele = = number of (+) alleles total number of alleles (both + and -) 2(# of +/+ students) + 1 (# of +/.00 71 .55 0. Population geneticists use the terms p and q to represent the frequencies of the (+) and (-) alleles. Table 2. Remember. a class of 32 students (N) will have a total of 64 (2N) instances of each locus.528 2.00 = 4. Genotypic Frequencies Category Homozygous (+/+) Heterozygous (+/-) Homozygous (-1-) Total for Alu in a USA Sample Frequency 2.050 10.21 Number = = 1.

Calculated Category Allelic Frequencies Number for USA Frequency p := (+) alleles (-) alleles i" . is one of the foundations of population genetics. the Hardy-Weinberg equation says thai = = = p2 = the expected frequency of the (+/+) genotype in the population 2pq = the expected frequency of the (+/-) genotype in the population q2 the expected frequency of the (-1-) genotype in the population = I ~!' It is important to understand that p2. Using the values for p and q that you calculated in Table 2 for your class population. These theoretical frequencies are calculated using the observed values for p and q. and they may not be realized in real-life population samples if one of the conditions is not met. I = 1. calculate p2. for a population to achieve this equilibrium. 6.00 := " I" / . q Total alleles := . The equation describes the frequencies of genotypes in a population thai is at "genetic equilibrium". and q2... what might be some of the reason(s) for the difference? 7. your class resembles a Hardy-Weinberg genetic equilibrium. p2 + 2pq + q2 1. calculate p2. where p + q 1. and there must be no migration of individuals into or out of the population. and the allelic frequencies p and q. It is the algebraic expansion 'of (p + q)2 1. the population must be quite large. this indicates that the popula. If your observed (actual) genotype frequencies are not the same as the expected values.. calculate the allelic frequencies for the USA data as you did for your class population in Table 2. Table 4. 2pq. Given these conditions. 2pq. Do they come out to be the same as the genotype frequencies that you found in Table 1? If they do. The Hardy-Weinberg theory states that. Using the values for p and q that you calculated in Table 4 for the USA population sample. and q2 are expected. the members must mate randomly and produce offspring with equal success. 5. Do they come out to be the same as the genotype frequencies that you found in Table 3? Does this USA-wide sample suggest that the population of the USA is in Hardy-Weinberg equilibrium? 72 . they mayor may not be the same as the observed genotypic frequencies such as those shown in Table 1. 2pq. or an excessive mutation converting one allele to another. theoretical genotype frequencies of a population under Hardy-Weinberg equilibrium conditions. meaning that the frequencies are stable from generation to generation. How do your actual class data for genotypic and allelic frequencies compare with those of the random sampling of the USA population? Would you expect them to malch? What reasons can you think of to explain the differences or similarities? I " The Hardy-Weinberg equation."' ! i I Now. tion is in Hardy-Weinberg genetic equilibrium. using the data above. If the observed and expected genotypic frequencies are the same. and q2.

2.I~m£: -- _ Lesson 1 DNA Template Preparation Focus Questions 1. IJYhatis needed from the cells for peR? ) 3. Why is it necessary to chelate the melal ions from solution during the boiling/lysis step at 1DOUC? What would happen if you did not use a chelaling agent such as the lnstaGene matrix? . What structures must be broken to release the DNA from a cell? 4. Why do you think the DNA is stored cold with the InstaGene matrix after boiling the samples? 49 .

j \Ii ~ ~r I . 4. 5.. How did Taq DNA polymerase acquire its name? J i . i· 56 . G. Why is it necessary 10 have a primer on each side of the DNA segment to be amplified? . •t Why are there nucleotides (A. :. .'.1 i :}. 2.of the master mix.. ' Describe the three main steps of each cycle of peR arnpliticafion and what reactions occur at each temperature . ·3. You may need to use additional paper and a drawing to explain your answer. i. Explain why the precise length target DNA sequence doesn't get amplified until the third cycle. it. T.I I I I . and what are their functions? " II . Lesson 2 peR Amplification Focus Questions '1.and C) in the master mix? What are the other components. .

What kind of controls are run in this experiment? Why are they important? Could others be used? 68 . Explain the difference between an intron and an exon.Lesson 3 Gel Electrophoresis of Amplified peR Samples Focus Questions 1.. W_hy do the two possible PCR products differ in size by 300 base pairs? 3. Explain how agarose electrophoresis separates DNA fragments. 2. Why does a smaller DNA fragment move faster than a larger one? 4.

Expel the saline back into the cup.' . Resuspend Ihe pellel by vortexing or flicking Ihe tube so that no clumps of cells rem~in. II's OK for LI small amount of saline « 50 ]11. refill your lube wtlh more of your oral rinse. "~. Using a 2-20 III adjustable-vokrme micropipet set to 20 pl. . Label one 1. You should see a match-head sized pellet of whitish cells at the bottom of the tube. 2. bioI your tube briefly on a paper lowel or tissue. aboullhe same size as your pellet) to remain in the bottom of Ihe tube. Being careful not to lose your pellet. A A § g 3. 8..Quick Guide Lesson 1 Cheek Cell DNA Template Preparation 1. V '. -. pour off the saline. ----. Centrifuge 5. and repeat Ihe spin. 6. 4. transfer all of your resuspended cells to the screwcap tube containing InstaGene. ~ ~ ~. 33 . Shake or vortex to mix the tube contents.1) l r 7. Label one screwcap tube containing 2001-11 of InstaGene matrix with your initials.-. Spin your tube in a balanced centrifuge at full speed for 2 minutes. . t: ~ ) ! " :. Obtain a cup containing saline solution from your instructor.5 ml micro test tube with your initials. carefully pour -1 ml of your saline rinse into your micro lest lube (use the graduations on the side of the micro lest tube to estimate 1 ml). If a P-1000 micropipet is not available.<. Screw the cap tightly on the tube. Transfer 1 ml of your saline rinse inlo Ihe micro test tube (NOT the screwcap lube) wilh your initials. remove your tube. Pour the saline into your moulh and rinse vigorously for 30 seconds. decant the saline. Aller peJleling your cells. When the centrifuge has completely stopped. If you don't see a pellet of this size.

and incubate at 56·C for 10 minutes in a water bath. 12. then place back in the 5S"C water balh for the remaining 5 minutes. place the tubes in the foam micro lest-tube holder. } _. . Incubate at 10a C lor 5 minutes. r cS '. Remove the tubes from the boiling water bath and shake or vortex the contents to resuspend. . shake or vortex.5 min 11..000 x g for 5 minutes (or 2. shake or vortex the lubes gently. D ----. Pellet the matrix by spinning at 6." t:::::J '1 r' ) I " ·1 Water both 100·C. Store your screwcap tube in lhe refrigerator until the next laboratory period (or proceed to step 2 of Lesson 2).. Remove the tubes.? / Centrifuge 34 'f0 .. ~ 11 56"C. and place the tubes in a boiling water bath (1 aO"C). ...~ I 9.---. When all members of your team have collected their samples. At the halfway point (5 minutes). 10 min Water bath ~ 1O.rr ---. ---...000 x g for 10 minutes) in a centrifuge.

Spin the screwcap lube for 2 minutes a16.Quick Guide Lesson 1. Transfer 20 IJI of the DNA template (the supernatant) from the screwcap lube lnto the bottom of the PCR tube. DNA template Supernatant "'MatriX '? 4. Place the PCR tube into the thermal cycler. Be very careful not to transfer any of the matrix beads into the PCR tube. Locate the tube of yellow master mix on ice and transfer 20 III of the master mix into lhe PCR lube. place the PCR tube in the capless tube as shown.000 x 9 (5 minutes at 2. The mixture should be yellow. 11 u PCR tuba Cilplosa tube 3. 2 PCR Amplification Obtain Ihe tube with your DNA template from the refrigerator. Centrifuge 2. Label a PCR lube and a capless micro test tube with your initials. 36 .' Mix by pipeUing up and down 2-3 limes.000 x g) in a cenlrifuge. The reactions will undergo 40 cycles of PCR amplification. Control reactions prepared by the instructor should also be placed into the PCR machine at this point. I~ MiI~tl!rmrx 5. and place both in the foam holder. Cap the PCR tube tightly.

5 !lL) (Wear gloves). Pour into assembledcasting stand and let set for 15 to 30 minutes. Pour an agarose mini gel. Connect lid to gelbox. To 50 mL of IX TAE buffer. add 0. . one per reaction per lane. 4. Electrophorese the samples at lOGY for 30 minutes or so. have the instructor load the standards and pcr reactions of 3 control samples (+/+.CHM329L Experiment il3 PCR part 2 (we elc it. 2. 3. and add EtdBr (2. Boil in microwave to dissolve agarose. Please record the identity of the samples and their corresponding location on the gel. Have the instructor help you photograph the gel to record your results. 3. Add 10 !JL loading dye to each per tube. Load your pcr reactions. Put gel into gelbox and cover completely with IX TAE buffer.2) 1".5 g agarose. and tum on power supply. cool to 65°C. +t-. In 4 of the wells. and -1-).

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