Purifying 5' phosphorylated primers for mutagenesis

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Fri Nov 27 09:22:45 EST 2009


I'm actually aiming to do a slight site-directed mutagenesis PCR reaction
shortly, I imagined it was as simple as doing a PCR with lower annealing
temperatures and a regular primer with a point mutation.. Am I mistaken?

What protocols do others on this list use for PCR-based mutagenesis? I'm
using KOD hotstart enzyme, which has exonuclease activity. It occurs to me
that it might inhibit any mismatches I might need for mutagenesis to occur,
should I be adding a solvent (such as DMSO) to inhibit exonuclease activity?

Thanks!
Cathal Garvey


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