In vitro mutagenesis

Vladimir Svetlov svetlov at oncology.wisc.edu
Tue Aug 26 09:14:08 EST 1997


In article <33FB756F.3039 at bcm.tmc.edu>, jorges at bcm.tmc.edu wrote:

> I would like to have any opinions on the various in vitro mutagenesis
> kits, especially the double stranded ones on the market. Tanks!

Promega pALTER/pSELECT works very well with both point mutations and
deletions of up to 200 bp long (never tried bigger ones), ds protocol is
quite effective - even with huge loop-outs no less than 50% of colonies
were mutants, but it requires subcloning and passage through a mutS host. 
Stratagene Qiuckchange is faster and works well with most medium-size
vectors, I've just used it to generate a deletion of a few dozen of bp -
worked like charm, just don't forget that each half of the big-deletion
primers in essense anneals independently. I did not find any "unspecified"
mutations in the rest of the clone. 
mutS host (P) and PCR (S) both are error-prone steps and should be taken
into consideration.
Regards,
V.



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