|Stock Reagent ||1x ||2x ||3x ||4x ||5x ||6x ||7x ||8x ||9x ||10x |
|25 mM MgCl2 (1.5mM final) ||1.5 ||3.45 ||4.95 ||6.45 ||7.95 ||10.5 ||12 ||13.5 ||15 ||16.5 |
|10x Buffer (-Mg) ||2.5 ||5.75 ||8.25 ||10.75 ||13.25 ||17.5 ||20 ||22.5 ||25 ||27.5 |
|1 mM ATP & nbsp; (100 mM final) ||2.5 ||5.75 ||8.25 ||10.75 ||13.25 ||17.5 ||20 ||22.5 ||25 ||27.5 |
|1 mM GTP (100 mM final) ||2.5 ||5.75 ||8.25 ||10.75 ||13.25 ||17.5 ||20 ||22.5 ||25 ||27.5 |
|1 mM TTP (100 mM final) ||2.5 ||5.75 ||8.25 ||10.75 ||13.25 ||17.5 ||20 ||22.5 ||25 ||27.5 |
|1 mM CTP (100 mM final) ||2.5 ||5.75 ||8.25 ||10.75 ||13.25 ||17.5 ||20 ||22.5 ||25 ||27.5 |
|OR -- for labelling DNA substitute above dCTP with: || || || || || || || || || || |
| 1 mM dCTP (cold) ||2.1 ||4.83 ||6.93 ||9.03 ||11.13 ||14.7 ||16.8 ||18.9 ||21 ||23.1 |
| 32P-dCTP (4µCi) ||0.4 ||0.92 ||1.32 ||1.72 ||2.12 ||2.8 ||3.2 ||3.6 ||4 ||4.4 |
|---------------------------- || || || || || || || || || || |
| Water ||0.7 ||1.61 ||2.31 ||3.01 ||3.71 ||4.9 ||5.6 ||6.3 ||7 ||7.7 |
|20 µM 5' primer (1 µM final) ||1.25 ||2.875 ||4.125 ||5.375 ||6.625 ||8.75 ||10 ||11.25 ||12.5 ||13.75 |
|20 µM 3' primer (1 µM final) ||1.25 ||2.875 ||4.125 ||5.375 TD> ||6.625 ||8.75 ||10 ||11.25 ||12.5 ||13.75 |
|Taq Polymerase (5U/µl) (1.5U) ||0.3 ||0.69 ||0.99 ||1.29 ||1.59 ||2.1 ||2.4 ||2.7 ||3.0 ||3.3 |
| SUM ||17.5 ||40.25 ||57.75 ||75.25 ||92.7 5 ||122.5 ||140 ||157.5 ||175 ||192.5 |
|DNA template: Q.S. with H20 to 7.5 µl || Dispense 17.5 µl of master mix per tube. Add 7.5 µl of DNA to bring final volume to 25 µl. Overlay with 50 µl of mineral oil. |
To reduce nonspecific amplification, increase the annealing temperature in 3 to 5°C increments. We have used the extension temperature without a separate annealing temperature for particular amplifications with good results.
"Hot starts" refer to the addition of the polymerase after the DNA has denatured. This should result in minimizi ng nonspecific priming at low temperatures. This is accomplished by heating the sample above the denaturing temperature and then adding the Taq DNA polymerase through the mineral oil layer into the aqueous solution containing the DNA. Wax plugs are also commercially available. They are melted above the sample (lacking polymerase) then allowed to harden at room temperature. Additional reagents can be added above the solid wax (i.e. Taq polymerase), and then the entire sample is added to the thermal cycle r. As the DNA is denatured and the wax melts, the enzyme transfers to the sample to begin amplification.
A typical cycling temperature profile is:
94°C for 1 minute
60°C for 1 minute
72°C for 1 minute
We use a 5 minutes time period at 94°C to denature template prior to the initial cycle. An old rule of thumb for a 72°C extension temperature is 1 minute for each 1000 base pairs of DNA being amplified. A 2 kb segment would need an extra minute, while segments under 200 bp don't need an extension plateau. More recently, Perkin Elmer Cetus has reported the extension rates for Taq, Stoffel and rTth polymerases are 2 - 4 kb per minute. Taq DNA polymerase is active over a broad range, not just at the extension temperature. A Perkin-Elmer representative said that they ha ve measured elongation rates of 75 bp/second at 70°C, 24 bp/sec at 55°C, and 1.5 bp/sec at 35°C. Thus, extension occurs throughout the annealing step, further stabilizing the primer-template interactions, but increasing nonspecific products. A final 5 minute incubation at 75#176;C is commonly added at the end of the cycles.
Cloning Amplified Products:
The ligation efficiency of amplified DNA varies from sequence to sequence. The cause of the problem is either addition of an A at the end of PCR products, and/or Taq polymerase enzyme remaining on the end of the DNA after amplification. Various solutions have been devised to compensate for these problems.
Perhaps the simplest strategy when you know in advance that you are going to clone the products is to design a restriction enzyme site(s) to the 5' end of the primers. Be sure these are unique to the primers and are not also present on the sequence you are amplifying. The products are then digested and subcloned into an appropriately digested vector. Note that some enzymes need a certain number of nucleotides beyond their recognition site for digestion.
In "Improved Cloning Efficiency of Polymerase Chain Reaction (PCR) Products after Proteinase K Digestion" by Crowe et al (1991) Nucl. Acids. Res. 19(1):184, the authors conclude that T aq polymerase sticks to the ends of amplified products. They found that proteinase K treatment increased the yield of their clones.
Vectors are commercially available that have an overhanging T on them to compensate for the potential overhanging A at the end of some PCR products. We have had many successful ligations of PCR products into blunt-end digested vectors, suggesting that there is a significant proportion of molecules that lack the overhang ing A residue. Others have treated their products for 15 minutes with 10 U of T4 DNA polymerase, or 30-60 minutes with 5-10 units of Klenow to produce blunt end products. For an alternate strategy, see Aslanidis and de Jong, Nucleic Acids Research 18:6069-6074. The method does not use ligation, and reportedly produces only recombinants with high efficiency. Small inserts can give light blue colonies when a pUC-based vector is employed.
; Mutation estimates vary from 10-3 to10-5 and vary with the length of the product and the number of cycles. Other parameters have also been found that influence this measurement. To maximize sequence fidelity of the products, or for use in random mutagenesis, the following table may be helpful.
|Component || Increased Fidelity ||Increased Infidelity |
|dNTP ||Equal concentrations of dNTPs @ 40-50µM each ||Unequal concentrations, 1-2 mM of 3 dNTPs with 0.1 - 0.2 mM of the fourth |
|Mg++ ||1 - 1.5 mM ||6 - 8 mM with 0.5 mM Mn ++ |
|Temperature ||increase ||decrease |
|[Taq] ||decrease ||increase |
|Time ||decrease extension time ||increase extension time |
|# cycles ||decrease ||increase |
Problem Solving Section:
++ ion concentration been titered? Reducing it may help.
- (1) No or few products detected
- Were all components added?
- Was the template denatured?
- Are the primers annealing and forming primer dimers?
- Are the cycling parameters correct, i.e. low enough for annealing and extension?
- (2) Non-specific product amplification
- Are the primers specific for the gene of interest?
- Was too much polymerase added?
- Should the annealing and/or extension temperatures be increased?
- Has the Mg
Reduce annealing/extension times, and/or decrease the number of cycles.
Was a non-template DNA negative control run?
- (3) Primer-dimer formation
- Are the 3' ends of the primers complementary?
- Increase the ratio of template to primers.
- Reduce the number of cycles.
- Increase the annealing temperature.
- (4) Poor restriction enzyme digests of PCR products
- The pH of the PCR buffer at 37°C is typically between 8.3 - 8.8, and may be higher than the pH optimum for most restriction enzymes. Promega advises to keep the PCR s olution below 50% of the digest volume.
Additional Notes on PCR Techniques:
Aliquots of amniotic fluid that have undergone multiple freeze-thaw cycles (which lyses cells) served as an adequate template.
McHale, R.H., P.M. Stapleton and P.L. Bergquist discussed amplification of DNA from tissue samples in "Rapid preparation of blood and tissue samples for polymerase chain reaction", Biotechniques 10: 20-23 (1991).
For RT-PCR reactions, Promega (Promega Notes 62:20, 1997) indicates that about 1 x 105 transcripts are needed to initiate the reaction. If the average mammalian cell has about 10 pg of RNA, 1 µg of total RNA should be fine for amplifying rare transcripts. AMV reverse transcriptase is active at 58°C, while M-MLV RT should not be used above 42°C.
Silver-stained DNA may be amplified as described by Raaphorst, et al, BioT echniques 20:78, 1996.
Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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Copyright 1996, 1997, and 1998 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Horowitz, R. and Frank, M. B. Polymerase Chain Reaction. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/pcr.html). 1997. Oklahoma City. Revision Date: July 6, 1998."