hot mutagenesis techniques

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Tue Nov 28 12:20:03 EST 1995


Mike Preston (preston at warren.med.harvard.edu) wrote:

:> 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.

Some years ago I had this same problem with the older verison of the `Altered
Sites' kit. I screened numerous transformants from many different experiments
by sequencing and never found the designed mutation. I gave up on it. FWIW,
the only kit component I found necessary to buy the kit is the repair primer,
which Promega will not give out the sequence of. Is anyone willing to disclose
their own designed ampicillin repair primer or the one that's used in the kit?

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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