site directed mutagenesis

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Thu Nov 12 12:49:05 EST 1998


At 16:22 11/11/98 -0600, Vladimir Svetlov wrote:
>The QuickChange kit from Stratagene works very efficiently (90%+ for point
>mutants, 50%+ for deletions of up to 1 kb and insertions of up to 100 bp).
>As there were some backorders with the kit we were forced to discover (we
>would never think of not buying the entire kit on our own...) that the
>method works fine with Pfu purchased separately from Stratagene or Promega
>and DpnI from just about anybody. Once I've used Pwo instead of Pfu and
>that worked too. The largest plasmid I ever used with this protocol was 7
>kb long. Let me know if you have specific questions about particular type
>of mutations you want to introduce. Overall this is by far the easiest and
>most efficient SD mutagenesis kit we have used.

Vladimir:

I "second" Vladimir's recommendation for Quick Change. For us, it has been
the cure-all for all of our SDM woes.  

I haven't pushed it much farther than Vladimir, only to about 7.7 kb.  The
number of recombinants goes down dramatically with increasing construct size.

One observation:  I would strongly recommend sequencing any mutants you
choose.  We have had the occurance that sometimes one or the other, or
both, oligos end up being incorporated into the mutant in, for lack of a
better term, a "tandem repeat".   For example, when using two opposite
36-mers, I've found upon sequencing that one of the oligo sequences picks
up a the end (either end) of one of the mutagenic oligo, thus throwing the
ORF out of frame.  It is possible that this may be due to our *not*
purifying the oligos by HPLC but we end up with so many recombinants all we
have to do is single sequence those that meet our initial screening criteria.

You couldn't pry this system from us!

Hope this helps,
David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
=============================



More information about the Methods mailing list