Stratagene's Quick Change Kit..Wow!

Bernard P. Murray, PhD bpmurray*STUFFER* at
Fri Feb 26 18:48:00 EST 1999

In article <2CCB6CA5A8A5D211AFD700A0240AFD6C613FE4 at>,
zje8 at (John R. McQuiston) wrote:

> Hi Everyone,
> I just used the Quick Change SDM Kit for the first time and for a large 
> mutation 6bp del. 2bp change.  I was in awe.  I screened 17 clones by 
> sequencing and EVERY single clone had the correct mutation.  I was
hoping for 1 
> or 2.  I would highly reccommend this procedure since it's as easy as setting 
> up a PCR reaction , a restriction digest and transformation.  
> I'm impressed, and also not affiliated with Stratagene in any way.
> John R. McQuiston
> Centers for Disease Control.

I don't think the efficiency of mutation has ever
been questioned (as long as the primer selection
is good).  I, too, find most/all clones have the
appropriate mutation when screened by restriction
site insertion/deletion but I am more concerned
that extra mutations have been introduced by Pfu
in the remainder of the construct (its "high"
fidelity not "perfect" fidelity).  I habitually
sequence the region surrounding the mutation
then chop it out and subclone it.

What length/proportion of your 17 clones did
you sequence?

Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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