Purifying 5' phosphorylated primers for mutagenesis

Duncan Clark via methods%40net.bio.net (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 
masterpiece:
>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?

No.

>
>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 
equivalent.

Duncan


-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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