site directed mutagensis

Jose de las Heras via methods%40net.bio.net (by josenet from tiscali.co.uk)
Tue Feb 12 19:59:54 EST 2008


"doaa zineldeen" <zineldeen from gmail.com> wrote in message 
news:mailman.587.1202621439.2451.methods from net.bio.net...
> hello
> I just want if anyone have a protocol for pcr mediated site directed
> mutagensis without using stratagene kit
> i have a template vector containing the insert and i want to mutate k81 
> into
> A i designed 2 primers flanking the mutation site and i want can i perform 
> 2
> pcr steps and a third mix with normal polymerase i am using phusion
> can i proceed to get the new product containing the mutated sites
> If someone can help i am really grateful
> Doaa

For a point mutation (or a couple) the "Quickchange" PCR system works well. 
I know you don't want to use Stratagene's kit... and neither do I. But you 
don't need any kit to use the strategy. Read their manual to get a few 
hints, and do it with your own reagents... I just sequenced 3 clones out of 
8 I picked for a double point mutation (vector+insert size = 6kb)... all 
three were perfect. Just one step.

For other things I use a "megaprimer" approach (google) which is another 
simple strategy.

Jose 




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