Stratagene's Quick Change Kit..Wow!
John R. McQuiston
zje8 at cdc.gov
Sat Feb 27 09:44:32 EST 1999
I sequenced 800 bp of a 1400bp ORF in both directions for all 17 clones. I'm
doing the expression experiment now. I was looking for a change in epitope at
that specific point so that was what was most important. After I get the
results of the expression I'm going to go back and reseq. the whole thing
of 1 or 2 just to make sure.
In article <bpmurray*STUFFER*-2602991548000001 at mac-daddy.ucsf.edu>,
>In article <2CCB6CA5A8A5D211AFD700A0240AFD6C613FE4 at mcdc-us-news.cdc.gov>,
>zje8 at cdc.gov (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
>> 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
>Bernard P. Murray, PhD
>Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
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