Wittrup: PCR

Materials

Taq polymerase (usually 5 U per L) and 10X buffer

MgCl2

forward primer

reverse primer

DNA template

PCR grade water

dNTP mix or each of dATP, dCTP, dGTP, dTTP

2.5 M betaine (optional)

DMSO (optional)

Mix the components of each 50 L reaction on ice:

reagent final concentration 10X buffer 1X MgCl2 final concentration 1.5 to 2.5 mM forward primer 0.5 uM reverse primer 0.5 uM dNTP 10 mM each of dATP, dGTP, dTTP, dCTP DNA template typically 10 ng water x L to 49 L final volume Taq Polymerase 2.5 to 5 Unit

DMSO and betaine tend to improve specificity of the annealing reaction. If the use of DMSO and betaine are desired, the final concentration of DMSO and betaine should be 3% (v/v) and 1 M.

Proceed with thermal cycling conditions outlined below: Denature 1 min at 94 C Anneal 1 min at the proper annealing temperature (depend on the primers) Extend 1 min for each 1 kb at 72 C Repeat for 30 cycles (typical amplification) Final extension for 5 to 10 min at 72 C.