Purifying 5' phosphorylated primers for mutagenesis
(by blackhole from abuse.plus.com)
Fri Nov 27 11:52:05 EST 2009
Historians believe that in newspost
<mailman.373.1259333775.1133.methods from net.bio.net> on Fri, 27 Nov 2009,
Cathal Garvey <cathalgarvey from gmail.com> penned the following literary
>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.
Phusion also works nicely.
> 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?
DMSO will do nothing to inactivate the proof-reading activity. You
actually want proof-reading activity, else your PCR product is going to
have errors where don't want them.
Look up Quikchange from Stratagene and use your KOD in your own homebrew
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
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