Polymerase Chain Reaction

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.

Procedure

1. DNA template: Between 1 and 5 ng of cloned DNA or between 40 and 100 ng of genomic DNA should be used per reaction. It is convenient to dilute template stocks to an appropriate concentration, e.g., 5 ng/µL in
   dH₂O for cloned DNA.
2. Primers: Primers should be prepared by butanol extraction and resuspended in dH₂O at 100 ng/µL. Each primer should be used at ~100 ng per reaction.
3. Buffer: Buffer should be prepared as a
   10X stock. 10X PCR buffer: 100 mM Tris. HCl pH 8.3, 500 mM KCl, 15 mM MgCl₂. This buffer can be prepared containing 0.1% gelatin.
4. Taq DNA polymerase: Taq should be used at 2.5 U per reaction. Amplitaq Polymerase (Perkin Elmer Cetus) is provided at 5 U/µL.
5. Magnesium: Extra magnesium can be added to the PCR reaction. If using the buffer above, a final Mg²⁺ concentration of 1.5 mM 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 10 mM. Magnesium can be prepared as MgCl₂ at 25 mM and autoclaved. Increasing the magnesium concentration has the same effect as lowering the annealing temperature.
6. Nucleotides: dNTPs should be prepared from 100-mM commercial stocks (Promega or Boehringer Mannheim) as a 10X stock at 2 mM each dNTP. This is most easily done by adding 2 mL of each dNTP to 92 mL dH₂O 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 distilled water is in doubt. It can be aliquotted into 1-mL volumes and kept separate from DNA and other sources of contamination. Each aliquot should be discarded following a single use.
8. Paraffin oil: In some instruments, paraffin oil must be added to prevent evaporation of the sample.