SDM kits

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Wed Sep 23 12:12:33 EST 1998


At 12:02 9/22/98 -0700, Dave Oliver wrote:
>I'm looking for information on any of the commercially available site
>directed mutagenesis kits.  I'm considering either clontechs
>"transformer" kit or promega's "altered site II" kit.   If anyone has
>used either of these kits (or others) successfully or unsuccessfully I
>would greatly appreciate your advice/reply.

Dave:

I think I can help.  I've used the "Transformer" system and as long as I
only needed to do one SDM, it worked fine.  Well, what I needed to do in
the assembly of a construct was to introduce a few new restriction sites
and eliminate others.  Unfortunately for me, I could only manage this once
per vector and I don't know why.  In this system, two oligos are required -
one to serve as a selection primer, the second to introduce the desired
site.  So to introduce 2 sites, required 4 oligos.  For some reason, even
though we CsCl purified the mutant plasmids between SDMs, we could only
effect one SDM.  We were unable to introduce a second restriction site into
the vector that had been previously altered.  I know, it doesnt' make any
sense - but we dinked with it for 2-3 months before calling it quits.

We then moved onto the Stratagene Quick-Change system and never looked
back.  The transformer is T4-pol based (no-PCR), the Quick-Change is Pfu
based (PCR).  It is obviously imperative to sequence your favorite
plasmid/construct for errors regardless.  We have had great success with
this system but it does come with a quirk making sequencing paramount.  As
it is PCR based, we have found our mutagenic oligos (which are exactly
matched and point in opposite directions) incorporated into the mutagenesis
process.  We have occasionally found the oligos incorporated either
upstream or downstream of the desired changes.  However, the recombinant
statistics are such that we have always had plenty of colonies to choose
from.  As a rule we'll pick 8-10 "mutants" that digest as desired and
sequence them all.

I 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.
=============================



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