Asymmetric PCR

Asymmetric PCR is used to produce single-stranded DNA from a template. If one PCR primer is vastly in excess compare to the pairing PCR primer, the majority of the PCR product will be the sense strand of the PCR primer in excess.

Make the following reaction mix:

10X PCR Reaction Buffer 5 ml
Primer 1 (20 mM, 20 pmol/ml ) 1 ml
Primer 2 (0.2 mM, 0.2 pmol/ml ) 1 ml
25 mM MgCl2 3 ml
10 mM dNTP (mix of 10 mM dATP, dCTP, dGTP, dTTP each) 1 ml
PCR DNA Polymerase (1 units/ml) 1 ml
DNA template (50-500 ng/ml) 1 ml
ddH2O 37 ml
Total volume 50 ml

See PCR for details and optimization.