Optimizing PCR Protocols
Standard reaction mixture:
50-100 ng genomic DNA1X reaction buffer (This can be the buffer provided with the DNA polymerase.)
1.5 mM MgCl2 final (optimal may need to be determined, range: 1.0-5.0 mM)
0.2 mM dNTPs final
0.4 µM each primer final (range: 0.2-1.0 µM)
0.25-0.5 units thermostable DNA polymerase per reaction
Melting temperature (Tm):
Melting temperatures for oligonucleotides can be calculated from simple formulae (e.g. Tm = 2[A+T] + 4[G+C]), or determined by software programs developed specifically for this purpose. It is unclear which is the best method to most accurately calculate Tm.
Optimal annealing temperature (Ta):
A general rule of thumb is to use a temperature approximately 5 °C lower than the Tm of the primers. Alternatively, there are more complex equations to calculate Ta (Rychlik et al., Nucleic Acids Research 18:6409-6412).
Additions that may improve results:
DMSO (up to 10%)Detergents (NP40, Triton X-100, Tween® 20) up to 1%
Perfect Match® DNA polymerase enhancer (Stratagene)
