Stratagene's Quick Change Kit..Wow!

[John R. McQuiston]

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.  

John




In article <bpmurray*STUFFER*-2602991548000001 at mac-daddy.ucsf.edu>, 
bpmurray*STUFFER*@socrates.ucsf.edu says...
>
>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 
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
>-- 
>Bernard P. Murray, PhD
>Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA