PCR Amplification and Cloning
of the Human Clotting Factor IX Gene.

A component of the Lycoming College Molecular Biology and Bioinformatics Project

Jeffrey D. Newman, Lycoming College © 1999

The Polymerase Chain Reaction (PCR) and cloning are two common ways to make many copies of a specific DNA sequence. An important concept is that PCR allows the specific amplification or production of a desired DNA fragment from a complex mixture of DNA sequences. In cloning, one inserts a specific DNA fragment into a plasmid or bacteriophage vector to create a recombinant DNA molecule. The vector contains the DNA sequences necessary for replication and maintenance in bacteria or yeast and can easily be purified away from the host’s chromosomal DNA.

The overall goal of this experiment is to amplify a small region within your own personal genome, insert this DNA into a plasmid vector and identify recombinant plasmids. The strategy to be used is a general one that could be applied to clone any gene or other relatively small (<10 kbp) DNA segment whose sequence is known. This strategy has become tremendously important now that many entire genomes have been sequenced and the human genome project has reached the large-scale rapid sequencing phase. This freely available data will revolutionize biomedical research and forever change the way we think about our species.

Timetable

Week 1 – Overview, isolate cheek cell DNA, set-up PCR, agarose gel

Week 2 – Clean up PCR product, cut PCR product and plasmid vector with restriction enzyme. Analyze products by agarose gel electrophoresis.

Week 3 - Ligate DNAs, Transform into E.coli.

Week 4 - Isolate Plasmid DNA from Transformants.

Week 5 - Restriction enzyme digestion of plasmids and agarose gel electrophoresis.

In addition to the experiments directly involved with achieving our goal, we will conduct a number of supporting activities as well. These activities are important for you to understand the theory behind the specific techniques.

 

 

Week 1 – Extraction of cheek cell DNA and PCR amplification of the clotting factor IX gene.

When one seeks to manipulate or study a particular region of DNA, it is useful to isolate and clone the DNA fragment of interest. In this experiment, we will amplify and clone a segment of the clotting factor IX gene.

 

The primers to be used in this PCR are as follows:

FIX exon7 HindIII 5'- ggtgaAAgcTTGATGCATTCTGTGGAGG -3'

FIX exon8 HindIII 5'- gtgagaagCTTCAGTAACATGGGGTCC -3'

Bases shown in lower case letters are not complementary to the target DNA, but were included to incorporate restriction enzyme recognition sites (AAGCTT) for HindIII. This common practice facilitates subsequent cloning of the PCR product. Because DNA is synthesized in the 5’ to 3’ direction, new nucleotides are always added to the free 3’ hydroxyl group of the deoxyribose sugar. Therefore it is the 3’ end of the primer where base pairing with the template is critical, while the 5’ end can be varied somewhat without negative effects.

The beauty of PCR is the ability to specifically amplify a desired piece of DNA from complex mixture of other DNA sequences. The human genome contains about 3,000,000,000 bp (3 x 109 bp), and we will amplify a specific 1182 bp fragment from that - Amazing! To hedge our bets, however, we will also amplify the fragment of interest from a plasmid clone (3.8 kbp) that contains this region.

  • Critical Thinking: Why are we more likely to get a PCR product using the plasmid as a template than when using genomic DNA as the template?
    •

    Use of Pipettors – Review and Reference

    Compared to many other areas of biology, the technical skills required to do molecular biology are very simple; the key is to get comfortable working with small volumes. These small volumes are measured out using micropipettors. A "set" consists of three pipettors that together can accurately measure and dispense volumes from 0.5 mL to 1000 mL (1 mL).

    Your lab instructor will demonstrate the use of the pipettors.

    1. Set dial to desired volume. Do not try to turn volume setting dial beyond the maximum volume for the pipettor. This is how students most frequently damage the pipettors.
    2. Attach a pipette tip. The small yellow or white tips are used on the I-20, I-100 and I-200’s; the large blue tips are used with the I-1000 pipettors. Be sure tip is attached securely to pipettor, liquid will drip out if seal is not tight.
    3. Press plunger down to 1st stop. Place tip into liquid
    4. Slowly release plunger so that liquid is drawn into pipette tip. Always hold pipettors with the shaft pointing down, especially when liquid is in the tip. If liquid does get into the shaft, please tell your instructor immediately, so that it can be cleaned out. Damage and corrosion can occur if salt solutions dry inside pipettor.
    5. Dispense liquid into desired tube by pushing down plunger to the second stop. Remove tip from liquid before releasing plunger, otherwise you will draw liquid back into tip.
    6. Eject used tips into coffee cans at your bench.

     

    Practice using the pipettors to measure the following volumes of water; 2 mL, 12 mL, 32 mL, 100 mL, 350 mL, 900 mL. How could you determine whether your measurements were accurate?

     

     

    A. Isolate Cheek Cell DNA for PCR Amplification:

    1. Pour all of the into your mouth and vigorously swish 25 mL saline solution for 10 seconds. Do not discard the empty tube that contained the saline solution.

    2. Expel the sample solution into a paper cup.

    3. Pour the sample solution from the paper cup back into the tube that contained the saline solution and close cap tightly.

    4. Spin sample in preparatory centrifuge on high speed for 10 minutes.

    5. Carefully pour off supernatant (liquid on top) into sink and place tube containing your cells on ice.

    6. Use 100-1000 µl micropipettor to transfer 500 µl Chelex solution to the tube containing your cell pellet:

    7. Set the micropipettor to 500 µl and pipet the Chelex solution in and out of the pipet tip several times to suspend the Chelex beads.

    8. Before the Chelex has a chance to settle, add the 500 µl to the centrifuge tube containing your cell pellet.

    9. Mix cells and Chelex by pipeting up and down several times until no visible clumps of cells remain.

    10. Using the same pipet tip, transfer 500 µl of your resuspended sample into a clean 1.5 ml tube. Be sure to label the cap of the tube with your initials.

    11. Place your tube in a boiling water bath for ten minutes.

    12. Carefully remove your tube from the boiling water bath and place on ice for one minute.

    13. Place your tube in a microcentrifuge (opposite someone else¹s tube!) and centrifuge for 30 seconds.

    14. Use a fresh pipet tip to transfer 200 µl of supernatant (clear solution on top) to another clean 1.5 ml tube. Label with your name.

    B. Set up PCR as follows:

    1. Prepare master mix for 2 x 50 m L reactions in a 0.5 mL microfuge tube. A master mix contains all of the common components for a set of reactions. In this case, the two PCRs will be identical except for the template DNA. Therefore, we can mix all of the components for the two reactions except for the template DNA, then divide them into two tubes for subsequent template DNA addition. Using master mixes serves several purposes.

    PCR Master Mix

    2. Distribute 47 mL master mix into labeled 0.5 mL microfuge tubes (gDNA-PCR, plasmid-PCR)

    3. Add 3 mL template DNA1 (5 - 20 ng for plasmid; 300 - 3000ng for genomic DNA).

    Critical Thinking: Why would an excess of target sequence cause problems with PCR?

    4. Add 50 mL mineral oil to prevent evaporation, close tubes tightly, store on ice until thermal cycler is ready to run.

    5. Store your genomic and plasmid DNA samples at –20oC for analysis next week.

    6. Initiate Factor IX thermal cycling program.

    Factor IX

    Notes:

    1 To improve specificity, template DNA concentration, annealing temperature and Mg2+ concentration may be varied.

    2 1 minute extension time should be used for each kbp of product expected.

     

    C. Prepare 1% agarose gel – To save time during next week’s lab, today we will prepare the agarose gel to analyze the PCR products, wrap it in plastic and store it until next week.

    1. Securely tape the ends of a gel tray such that a small amount of tape is on the underside of tray. Place tray on a sheet of plastic wrap in case of spillage. Align comb in tray parallel with and 1-2 cm from the end of the tray.

    2. Add an appropriate amount of agarose (0.4g) to 40 mL water in a 125 mL erlenmeyer flask, heat mixture in microwave on high setting for 45 sec - 1.5 min, or until mixture begins to boil.

    3. Using a folded paper towel to hold the neck of the erlenmeyer flask, swirl the gel mixture well, and return to microwave. Heat for an additional 30 - 45 sec, or until mixture begins to boil.

    4. Carefully remove molten gel solution from microwave using a folded paper towel to hold the neck of the erlenmeyer flask.

    5. Add 0.8 mL 50x TAE buffer, 12 m L 1.6 mg/mL ethidium bromide (final conc = 0.5 m g/mL), swirl to mix, pour into gel tray, allow to stand at room temp for 20 - 30 min to solidify.

    6. Remove comb, slide gel off tray onto plastic wrap, wrap gel, store at 4oC.

     

     

    Week 2 – Clean up PCR product, cut PCR product and plasmid vector with restriction enzyme. Analyze products by agarose gel electrophoresis.

    Our goal for today is to prepare the DNA samples for the ligation reaction to be conducted next week. This preparation involves cutting the PCR product and pBS vector with the restriction enzyme HindIII to generate "sticky ends" and estimating the concentration of the DNA.

    The PCR mix contains components that would inhibit the restriction enzyme digestion, therefore we must purify the PCR products using the QiaQuick spin column. The column contains a DNA-binding membrane that captures the DNA from the PCR mixture, while allowing oligonucleotide primers, buffer components, and enzyme to pass through. After washing the membrane, the DNA can be eluted in a buffer more appropriate for restriction enzyme digestion.

    A. Purification of PCR product.

    1. Transfer 40mL of both PCR mixtures to a single new 1.5 mL microfuge tube, save the remaining 10mL of each for analysis by gel electrophoresis in step C.

    2. Add 400mL buffer PB to combined PCR samples, transfer this mixture to a spin column nestled inside a 2 mL microfuge tube.

    3. Centrifuge for 1 min., discard filtrate, put spin column back into tube.

    4. Add 750 mL buffer PE to spin column, centrifuge for 1 min., discard filtrate, put spin column back into tube.

    5. Centrifuge again for 1 min., discard 2 mL tube containing filtrate, place spin column in new 1.5 mL microfuge tube labeled "purified PCR".

    6. Pipette 30 mL buffer EB directly onto filter (but do not touch filter with pipette tip), let it stand for 2 min, centrifuge for 1 min. Discard spin filter, place tube containing eluted DNA in ice bucket.

    B. Restriction enzyme digestion

    Restriction enzymes recognize specific DNA sequences and cut the DNA at those sequences. Some enzymes, such as EcoRV, cut both strands opposite each other and create blust ends. Other enzymes, such as HindIII create staggered cuts that leave single stranded overhangs referred to as sticky ends. Base pairing of the complementary sticky ends on separate DNA molecules holds them together so the enzyme DNA ligase can connect them together to form a recombinant DNA molecule.

     

    EcoRV

    HindIII

    5'... G A T A T C ... 3'

    3'... C T A T A G ... 5

    5'... A A G C T T ... 3'

    3'... T T C G A A ... 5

    5'... G A T

    3'... C T A

    A T C ... 3'

    T A G ... 5’

    5'... A

    3'... T T C G A

    A G C T T ... 3'

    A ... 5'

     

    It is critical that the volumes of solutions used in this exercise be accurate. If you are not confident in your pipetting skills, please ask for help! All tubes should be labeled on their side with the contents, and on the top with your group designation.

    1. Label the sides of three 0.5 mL microfuge tubes with the following designations:

    gDNA + HindIII, PCR + HindIII, pBS + HindIII

    2. Set up master mix in "MM" tube as follows.

    3. Distribute 5 mL of master mix into each of the 3 HindIII tubes, discard MM tube.

    4. Set up restriction digests (20 mL total) by adding 15 mL of purified PCR product; pBS stock (50 ng/mL); gDNA to appropriate tubes. Incubate the tubes in the 37oC heating block for 45 min.

    C. Gel electrophoresis

    1. Fill gel chamber with 1x TAE buffer such that the level of liquid just covers center platform. Remove tape from ends of gel, place gel in chamber with the wells near the negative electrode (anode-black), add sufficient 1x TAE to just cover the gel.

    2. Cut a small piece of parafilm, place on bench near gel, "spot" a 1-2 mL aliquot of loading dye onto parafilm for each sample to be loaded on gel.

    3. Draw sample into a pipette tip, pipette up and down onto a spot of loading dye to mix, load sample into well of gel. Be careful not to poke pipette tip through bottom of well. Samples should be loaded in the following order (from left right):

    4. Place cover over gel chamber, turn on power supply and set to 100 V, press run button. Confirm proper operation by checking for gas production (bubbles) at electrodes (electrolysis of water).

    5. Heat remaining 15 m L of PCR + HindIII and pBS + HindIII in 70oC heating block for 20 min to inactivate enzyme. Centrifuge briefly to collect contents in bottom of tube and store in freezer box at –20oC.

    6. After fastest migrating blue dye (bromophenol blue) has migrated 2/3 the length of the gel, turn off power, carefully remove gel from chamber, drain, slide onto piece of plastic wrap. Photograph the gel under UV light.

    7. After completing exercise D below (while gel is running), estimate the DNA concentration of your HindIII cut vector and PCR product by comparison of the band intensity to bands containing a known amount of DNA.

     

    D. Estimating the amount of DNA in a band on an agarose gel - the concept.

    1. Measure the length of the piece of rope at your benchtop (it should be 50 cm). Determine the mass of the rope and enter it in Table 1 on the next page.

    2. Cut the piece of rope in half (two 25 cm pieces) and determine the mass of one piece, enter it in Table 1. Put the weighed piece aside temporarily.

    3. Cut a 1 cm piece of rope from the unweighed 25 cm half of rope. How much do you think it weighs? Enter your prediction in Table 1. Weigh the 1 cm piece, enter the mass in Table 1. How close was your prediction?

    4. Complete Table 1 by predicting the mass of 2.5 cm, 4.5 cm, 6 cm, and 11 cm pieces of rope, cutting these from the unweighed 25 cm half of rope (now 24 cm), and determining the mass of each piece.

    5. Total the masses of all the pieces 25 cm and smaller, do these add up to the mass of the original 50 cm piece of rope?

    6. If you had three 50 cm pieces of rope, what would be their combined weight? If you then cut a 1 cm segment from each rope, how much would the 1 cm pieces weigh (together)? What fraction of the total weight is this?

    7. Suppose you did not know how many pieces of rope you had, but did know that they were all 50 cm long and together weighed 1000g. If a 1 cm piece was cut from each rope, and the 1 cm pieces were weighed together, what would their mass be?

    8. The phage lambda genome is approximately 50 kb. If 1000 ng (1 m g) of lambda DNA was cut to release a 1 kb fragment, what would be the mass of DNA in the 1 kb fragment?

    9. When lambda DNA is cut with the restriction enzyme BstEII, the fragments listed in Table 2 are produced. If 1000 ng of lambda DNA was cut, how much DNA would be present in the bands corresponding to each fragment?

    10. After photographing your gel, compare the staining intensity of your HindIII-cut PCR product and HindIII-cut pBS vector to the bands of your l +BstEII standard. Since the amount of DNA in each of those bands is known, you can estimate the concentration of your two DNA samples to be used for cloning next week.

    Table 1. Relationship between rope length and mass.

    Length of rope (cm)

    Actual mass (g)

    Predicted mass (g)

    50 cm

    		    

    25 cm

    		    

    1 cm

    		    

    2.5 cm

    		    

    4.5 cm

    		    

    6 cm

    		    

    11 cm

    		    

    Total

    		   

     

     

     

    E. Determining the size of DNA fragments on a gel.

    The BstEII-cut lambda DNA was used as a standard for size as well as for amount. Measure the distance migrated by each DNA fragment by measuring from the front (leading edge) of the well to the front of each band and enter the data in Table 2. The migration of linear DNA through a gel shows an inverse, exponential relationship with its size (mass, length). On the graph paper provided, plot three graphs, (using your own data) each with migration on the X-axis:

    Draw a straight line through the data points on graphs b and c (points at the extremes probably will not fall on the line due to a limited range of effective separation by agarose and measurement errors). These are your standard curves. Measure the distance migrated by the PCR product and pBS vector, locate these distances on your standard curve, estimate the sizes of these DNAs based on the standard curve.

     

    Table 2. Amount and migration of DNA bands after BstEII-digestion of 1000 ng of lambda DNA.

    Amount of DNA (ng) Length of Fragment Log of length Distance Migrated (mm)
      8454 bp 3.93  
      7242 bp    
      6369 bp    
      5686 bp    
      4822 bp    
      4324 bp    
      3675 bp    
      2323 bp    
      1929 bp    
      1371 bp    
      1264 bp    
      702 bp    
      224 bp    
      117 bp    

     

    Week 3 - Ligate DNAs, Transform into E.coli.

    A. Ligation

    1. Assemble the following ligation reaction (15 m L) in a 0.5 mL microfuge tube labelled "ligation":

    2. To dissociate sticky ends, heat mixture in 70oC heating block for 2 minutes, tap spin, place tube in ice.

    3. Add 1 mL T4 DNA ligase, mix by pipetting, incubate at room temp. (20 - 25oC).

    4. After 1 hr, transform half of the ligation (7.5 mL) into an appropriate bacterial strain. Allow the other half to incubate at room temp. overnight.

    B. Preparation of competent cells.

    1. Inoculate bacteria to be transformed (E.coli strain TB1) into 6 ml LB liquid, incubate in shaker at 37oC, 250 rpm for 2.5 - 3.5 hrs. (until culture becomes slightly turbid). (already done for you)

    2. Centrifuge culture at setting 5 or 6 of the clinical centrifuge for 5 min., decant supernatant, add 4 - 7 mL ice cold sterile 0.1 M CaCl2, vortex to resuspend cells, place on ice for 30 min.

    3. Centrifuge cells in cold room at setting 5 or 6 of the clinical centrifuge for 5 min., decant supernatant, add 0.5 mL ice cold sterile 0.1 M CaCl2, vortex to resuspend cells, place on ice.

    C. Transformation of cells with ligation.

    1. Dilute 7.5 mL ligation into 100 m L 0.1 M CaCl2 in a labeled sterile 1.5 mL microfuge tube, place on ice.

    2. Add 100 mL competent cells to chilled tube containing DNA, mix gently, incubate on ice for 30 min.

    3. Heat shock cells in 42oC heat block for 2 min., add 1 mL LB liquid, mix by inversion, lay on side in 37oC incubator, incubate for 1 - 3 hrs.

    4. Centrifuge cells briefly (~15 sec.), decant most of supernatant, resuspend cells in remaining supernatant by vortexing, spread entire mixture onto LB Amp, X-gal plate. Incubate plate at 37oC for 16-20 hrs.

    transformation notes:

    A. It is very important that bacteria be harvested while still in the log phase of growth, and be kept ice-cold during the entire procedure.

    B. If screening for the presence of an insert via a -complementation, spread 50 mL X-gal (5-bromo-4-chloro-3-indolyl b -D-galactopyranoside) (20 mg/mL in dimethylformamide (DMF)) and 5 m L IPTG (isopropylthio-b -D-galactopyranoside) (100 mg/mL in H2O) onto plate before step 6. IPTG may be omitted when transforming into E. coli strain TB1.

    5. After colonies have grown up for 14-18 hrs, pick 1 blue and 6 white colonies with toothpicks, place the toothpick into labeled and numbered tubes containing 2 mL LB + ampicillin liquid. Store tubes in the refrigerator. These tubes will be placed in the 37oC incubator-shaker the day before you are to isolate plasmid from the culture.

     

     

     

    Week 4 - Plasmid DNA minipreps - CTAB method

    Theory: The cationic detergent cetyltrimethylammonium bromide (CTAB) forms an insoluble complex with DNA at low [NaCl], while proteins and polysaccharides remain in solution. At higher [NaCl], the CTAB becomes soluble allowing ethanol precipitation of pure Na-DNA.

    Procedure:

    1. Fill a 1.5 mL microfuge tube with an O/N culture of E. coli containing the desired plasmid.

    2. Centrifuge tubes for 30 sec-1 min; decant supernatant into waste can; tap tube containing cell pellet on paper towel to remove any remaining liquid.

    3. Resuspend cell pellets in 175 m L STET buffer by vortexing.

    4. Add 12.5 m L lysozyme solution, vortex briefly, place in boiling water bath for 1 min.

    5. Immediately spin for 10 min. in microfuge, use a toothpick to remove pelleted cell debris from the supernatant. Discard the pellet, but save the supernatants.

    6. Add 10 m L RNaseA, vortex briefly, incubate for 10 min. at 68oC.

    7. Add 10 m L CTAB solution, vortex briefly, leave at room temp for 3 min.

    8. Centrifuge at room temp for 5 min., carefully remove supernatant with a pipette and discard supernatant, save the pellets.

    9. Resuspend pellets in 300 m L 1.2 M NaCl by vortexing, 10. Add 750 m L ice cold 95% EtOH, vortex, place on ice for 15 min (or store at -20oC overnight).

    10. Centrifuge for 10 min. to pellet DNA. Discard supernatant. Save the pellet.

    11. Rinse pellet by adding 500 m L 70% EtOH, close cap, swirl ethanol around inside of tube to wash walls, decant well over a paper towel. Be careful not to lose DNA pellet.

    12. Dry DNA in speed-vac, resuspend in 50 m L TE buffer. Store at -20oC.

    Reference: Del Sal G, Manfioletti G, Schneider C (1989) BioTechniques 7:514-520 The CTAB-DNA precipitation method: A common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing.

     

    Week 5 - Identification of clones containing PCR product insert.

    1. Design and prepare a master mix (in a 0.5 mL microfuge tube) for restriction enzyme digestion of 5 m L of each plasmid with 1 m L PstI in a total volume of 10 m L. Distribute the master mix into labeled 0.5 mL microfuge tubes, add 5 m L of the appropriate DNA sample, incubate tubes in 37oC heating block for 45 min.

    2. Prepare a 1.2% agarose gel.

    3. Run restriction enzyme digestions on gel along with 10 m L l +BstEII marker. Photograph gel.

     

     

    Week 1 - equipment/materials/prep

    Equipment

     

    Materials

    One per class

    for every 2 pairs

    for every pair of students

     

     

    Week 2 - equipment/materials/prep

    Equipment

     

    Materials

    One per class

    for every 2 pairs

    for every pair of students

     

    Week 3 - equipment/materials/prep

    Equipment

    Materials

    One per class

    for every 2 pairs

    for every pair of students

    Week 4 - equipment/materials/prep

    Equipment

    Materials

    for every 2 pairs

    (100 mL = 8.0 g Sucrose + 0.1 mL Triton X-100 + 10.0 mL 0.5M EDTA pH 8.0 + 5.0 mL 1.0M Tris-HCl pH 8.0 + 84.9 mL dH2O)

     

    for every pair of students

    Week 5 - equipment/materials/prep

    Equipment

     

    Materials

    One per class

    for every 2 pairs

    for every pair of students

     

    This page was created or last modified on 10/18/99 by Jeff Newman
    and has been accessed  times since 10/18/99.

    Assistant Professor Web page: http://lyco.lycoming.edu/~newman
    Department of Biology Email: newman@lycoming.edu
    Lycoming College Phone: 570-321-4386
    Williamsport PA 17701 Fax: 570-321-4073

    © 1999  Jeffrey D. Newman