hot mutagenesis techniques

Mike Preston preston at warren.med.harvard.edu
Fri Nov 24 08:41:35 EST 1995


> varyc.mmcri at mmc.org wrote:
> : Hi all,
> : I've recently cloned a protein that binds (with differing affinities)
a family of structurally related ligands. I have the sequence in pMalC2
(expression) and pGemT (original
> : cloning from PCR). I'd like to subject the sequence to random discrete
mutagenesis in order to obtain a family of related proteins that possess
altered  relative
> : specificities and affinities for the family of ligands. 
> : Is there one or mure easily executed mutagenesis techniques that
people might recommend? I have a decent screening assay.
> 
> : Thanks in advance for any help.
> 
> : Cal Vary
> 
> Try the Promega's *Altered site II mutagenesis system*.
> It makes use of a knocked out ampicilin gene and a functional tetra gene.
> Once you have cloned your gene of interest in pALTER, you make it single 
> stranded. You then anneal the oligo (mutated primer and make sure it's 
> phosphorylated) to your gene. At the same time you anneal your amp repair 
> primer to teh vector -> Synthesize the 2nd strand with using T4DNA POl and
> then ligate using T4Ligase. The ds vector should have the mutated 
> nucleotide(s) in it.
> 
> Cheers
> Shahram Mori

Shahram,
   Our lab and the lab next door both tried to do a simple mutagenesis of
a restriction site and failed miserably using the Altered Sites II
mutagenesis kit.  It sounded easy and the theory behind it sound.  In
practice it was a loser (or perhaps we are just incompetent).  Tips from
Promega's Tech service did not help.  I finally went to sequence overlap
extension PCR and got the mutation first whack.  Have you used the Altered
Sites II kit?  If so can you provide the Net world with any tips for
success?  I would like to know if this expensive waste of freezer space is
worth it.

Mike Preston

-- 
Michael Preston
Channing Laboratory
preston at warren.med.harvard.edu



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