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Contamination of reactions is an often encounted problem with PCR. It is best avoided by using dedicated reagents, Gilsons, and plugged tips. Setting up a PCR reaction in a different location to where template or products are worked with is also a good precaution.
Reagents
1. DNA template: Between 1 and 5ng of cloned DNA or between 40 and 100ng of genomic DNA should be used per reaction. It is convenient to dilute template stocks to an appropriate concentration, eg 5ng/ul in dH20 for cloned DNA.
2. Primers: Primers should be prepared by butanol extraction (see appropriate method) and resuspended in dH20 at 100ng/ul. Each primer should be used at ca. 100ng per reaction.
3. Buffer: Buffer should be prepared as a 10x stock.
10x PCR buffer:
100mM Tris.HCl pH 8.3
500mM KCl
15mM MgCl2
This buffer can be prepared containing 0.1% gelatin.
4. Taq DNA polymerase: Taq should be used at 2.5U per reaction. Amplitaq Polymerase (Perkin Elmer Cetus) is provided at 5U/ul.
5. Magnesium: Extra magnesium can be added to the PCR reaction. If using the buffer above, a final Mg2+ concentration of 1.5mM will be obtained. If necessary, magnesium can be titrated to obtain an optimal concentration. Suggested concentrations for this would be 1.5, 3.0, 4.5, 6.0 and 10mM. Magnesium can be prepared as MgCl2 at 25mM and autoclaved. Increasing the magnesium concentration has the same effect as lowering the annealing temperature.
6. Nucleotides: dNTPs should be prepared from 100mM commercial stocks (Promega or Boehringer Mannheim) as a 10x stock at 2mM each dNTP. This is most easily done by adding 2ul of each dNTP to 92ul dH20 in an eppendorf.
7. Water: Water should be autoclaved and used solely for PCR. Milli-Q water is fine for PCR or "water for injection" if the milli-Q water is in doubt. It can be aliquotted into 1ml volumes and kept separate from DNA and other sources of contamination. Each aliquot should be discarded following single use.
8. Parrafin Oil: In some instruments parrafin oil must be added to prevent evaporation of the sample.
Reaction
1. The reaction is divided into three repeated cycles:
a) Denaturation 95¡C
b) Annealing 50¡C to 65¡C
c) Extension 72¡C
2. An appropriate annealing temperature must be determined for each pair of primers and template. Reactions should be carried out at a number of different annealing temperatures to determine an optimum value. Denaturation, annealing and extension times need to be determined on a per reaction basis also. Values for these are dependent on the PCR machine being used and are typically around 30 seconds for denaturation and 1 to 2 minutes each for annealing and extension for HFI machines PE 480, 9600). The number of cycles in each reaction is usually 25-40.
3. An extra denaturation step can be included prior to the reaction, this is usually 4 minutes at 95¡C. A "hot start" PCR has Taq polymerase added subsequent to this initial denaturation.
4. A typical reaction may be: Template (5ng/ul) 1ul 1ul
Primer A (100ng/ul) 1ul 1ul
Primer B (100ng/ul) 1ul 1ul
dNTPs (10x, 0.5mM) 5ul 10ul
Taq (5U/ul) 0.5ul 0.5ul
Buffer (10x) 5ul 10ul
dH20 36.5ul 76.5ul
50.0ul 100.0ul