The precise conditions under which PCR always works best are not known, and it seems reasonable to assume that no such standard conditions will be discovered.
The success of the PCR reaction depends on the kinetic advantage that high concentrations of primers have over relatively low concentrations of product strands. The optimal primer to template ratio is very important. If the ratio is too high, primer-dimers are formed. If the ratio is too low, products will not accumulate exponentially, since newly synthesized strands will renature after denaturation.
Reaction volumes of between ten to 100 microliters are generally used. The selection of times, temperatures, and number of cycles depends on the DNA being amplified and the primers chosen.
A sample protocol for PCR (the target region is about 1kbs) are below:
A) Equipment and reagents:
- Thermal cycler
- cDNA library from Lupinus luteus infected by Rhizobium c=7ng/l
- Expand High Fidelity PCR System (from Boehringer Mannheim)
- primers (10 M, melting temperature 66 and 65 C)
- dNTPs (10mM)
B) Reaction mixture: