Avoiding ContaminationPCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.
- DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas.
- A Laminar Flow Cabinet equipped with a UV lamp is recommended for preparing the reaction mixture.
- Fresh gloves should be worn for each PCR step.
- The use of dedicated vessels and positive displacement pipettes or tips with aerosol filters for both DNA sample and reaction mixture preparation, is strongly recommended.
- The reagents for PCR should be prepared separately and used solely for this purpose. Autoclaving of all solutions, except dNTPs, primers and Pfu DNA Polymerase is recommended. Solutions should be aliquoted in small portions and stored in designated PCR areas. Aliquots should be stored separately from other DNA samples.
- A control reaction, omiting template DNA, should always be performed, to confirm the absence of contamination.
These are only rough guidelines. Detailed instructions about PCR laboratory setup and maintenance may be found in PCR Methods and Applications, 3, 2, S1-S14, 1993.
Preparation of Reaction MixtureTo perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs, primers and template DNA in a single tube, which can then be aliquoted into individual tubes. Pfu DNA Polymerase should be added last. This method of setting reactions minimizes the possibility of pipetting errors and saves time by reducing the number of reagent transfers.
Reaction Mixture Set Up
Gently vortex and briefly centrifuge all solutions after thawing.
- Keep solutions on ice.
- Add to a thin-walled PCR tube, on ice:
Reagent Final
concentrationQuantity, for 50 µl
of reaction mixtureSterile deionized water - variable 10X Pfu buffer with MgSO4* 1X 5 µl 2 mM dNTP mix 0.2 mM of each 5 µl Primer I 0.1-1 µM variable Primer II 0.1-1 µM variable Template DNA 50 pg-1 µg variable Pfu DNA Polymerase 1.25 u/50 µl 0.5 µl * If using 10X PCR buffer without pre-added MgSO4, refer to the table below to determine required volume of 25 mM MgSO4 solution (for 50 µl total volume).
Final concentration of MgSO4 in 50 µl reaction mix, mM 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0 Volume of 25 mM MgSO4, µl 2 2.5 3 3.5 4 5 6 8
- Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube.
- If using a thermal cycler without a heated lid, overlay the sample with a half volume of mineral oil or add an appropriate amount of wax.
- Place samples in a thermal cycler preheated to 95°C and start PCR.
Recommended thermal cycling conditions:
Step Temperature, °C Time, min Number of cycles Initial denaturation 95 1-3 1 Denaturation 95 0.5-2 Annealing 37-70 0.5-2 25-35 Extension 70-75 2-4 Final extension 70-75 5 1 Components of the Reaction Mixture
Template DNA.
Usually amount of template DNA is in the range of 50 pg - 1 ng for plasmid or phage DNA and 0.11 µg for genomic DNA, for a total reaction mixture of 50 µl. Higher amounts of template DNA usually increase the yield of nonspecific PCR products, but if fidelity of synthesis is crucial, maximal allowable template DNA quantities in conjunction with limiting number of PCR cycles should be used to increase the percentage of "correct" PCR products. Nearly all routine methods are suitable for template DNA purification. Although even trace amounts of agents used in DNA purification procedures (phenol, EDTA, Proteinase K, etc.) strongly inhibit Pfu DNA Polymerase, ethanol precipitation of DNA and repetitive treatments of DNA pellets with 70% ethanol is usually effective in removing traces of contaminants from the DNA sample.
Primers.
PCR primers are usually 20-30 nucleotides in length. Longer primers provide sufficient specificity.
- The GC content should be 40-60%. The C and G nucleotides should be distributed uniformly throughout the primer. More than three G or C nucleotides at the 3'-end of the primer should be avoided, as nonspecific priming may occur.
- The primer should not be self-complementary or complementary to any other primer in the reaction mixture, in order to avoid primer-dimer and hairpin formation.
- The melting temperature of flanking primers should not differ by more than 5°C, so the GC content and length must be chosen accordingly.
- All possible sites of complementarity between primers and the template DNA should be noted.
- If primers are degenerate, at least 3 conservative nucleotides must be located at the primer's 3'-end.
- Estimation of the melting and annealing temperatures of primer:
- If the primer is shorter than 25 nucleotides, the approx. melting temperature (Tm) is calculated using the following formula:
Tm= 4 (G + C) + 2 (A + T)
G, C, A, T - number of respective nucleotides in the primer.
If the primer is longer than 25 nucleotides, the melting temperature should be calculated using specialized computer programs where the interactions of adjacent bases, the influence of salt concentration, etc. are evaluated. Optimal annealing temperature is generally 5°C lower than the melting temperature of the primer-template DNA duplex.- The 3'=>5' exonuclease activity associated with Pfu DNA Polymerase may degrade the primers. It is therefore important that Pfu DNA Polymerase be added last to the reaction mixture. Degradation of primers can be efficiently prevented by the introduction of phosphorothioate bonds at their 3'-termini (1). The use of longer primers with maximized GC content can be advantageous.
MgSO4 Concentration.
Pfu DNA Polymerase prefers MgSO4 to MgCl2. Since Mg2+ ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgSO4 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products. Optimal MgSO4 concentration is in the range of 2-4 mM. Increasing MgSO4 concentration from 2 to 10 mM does not vary significantly the error rate. If the DNA samples contain EDTA or other chelators, the MgSO4 concentration should be raised proportionally.
dNTPs.
The concentration of each dNTP in the reaction mixture is usually 200 µM. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even a single dNTP dramatically increases the misincorporation level. dNTPs concentrations of 100-250 µM of each dNTP result in the optimal balance of product yield (greater at higher dNTP concentration) versus specificity. In addition, the optimal concentration of dNTPs should be selected empirically.
Fermentas PureExtreme® dNTP Mixes, 2 mM (#R0241/2) and 10 mM (#R0191/2), are conveniently formulated for direct use in PCR.Pfu DNA Polymerase.
The concentration of the enzyme required for optimal PCR product yield and specificity depend on target to be amplified and the presence of inhibitors in the reaction mix (e.g., if the template DNA used is not highly purified). The optimal enzyme concentration is 1.25-2.5 u / 50 µl.
- 3'=>5' exonuclease activity associated with Pfu DNA Polymerase may degrade primers. It is important to keep PCR mixes and tubes on ice and add the enzyme to the reaction mixture last.
- Pfu DNA Polymerase has no detectable reverse transcriptase activity.
Reaction Overlay.
If necessary, the reaction mixture can be overlaid with mineral oil or paraffin (melting temperature 50-60°C) of special PCR grade. One-half of the total reaction volume is usually sufficient.
Temperature CyclingAmplification parameters depend greatly on the template, primers and amplification apparatus used.
Initial Denaturation Step.
The complete denaturation of the DNA template at the start of the PCR reaction is of key importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3 min at 95°C if the GC content is 50% or less.
For GC-rich templates the denaturation interval should be prolonged up to 10 min.Denaturation Step.
Usually denaturation for 0.5-2 min at 94-95°C is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. The GC content should be taken into consideration. Denaturation time can be optimized empirically.
Primer Annealing Step.
Usually the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2 min is usually sufficient. However, if non-specific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 1-2°C.
Extending Step.
Usually the extending step is performed at 70-75°C. Pfu DNA Polymerase exhibits lower extension rate than Taq DNA Polymerase, so 2 min extention time is recommended for every 1 kb to be amplified.
Number of Cycles.
The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. In most cases, 25-35 cycles are sufficient. In general, we suggest using the lowest possible number of cycles to achieve acceptable yield of PCR product and to lower a background of non-specific product.
Final Extending Step.
After the last cycle, the samples are usually incubated at 72°C for 5-15 min to fill-in the protruding ends of newly synthesized PCR products.
- Pfu DNA Polymerase, unlike Taq DNA Polymerase, lacks terminal transferase activity and generates blunt-ended PCR products, which can be used directly for blunt end ligation without any pretreatment of the ends.
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Updated gegužės 10, 2007 10:36
