Perplexing result with site-directed mutagensis

I-Jen Huang gr8bn at cc.usu.edu
Mon Nov 21 12:14:21 EST 1994


In article <3ajtdg$5jf at dartvax.dartmouth.edu> Donald.L.StGermain at dartmouth.edu (Donald L. StGermain) writes:
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>Subject: Perplexing result with site-directed mutagensis
>Message-ID: <3ajtdg$5jf at dartvax.dartmouth.edu>
>From: Donald.L.StGermain at dartmouth.edu (Donald L. StGermain)
>Date: 19 Nov 1994 04:02:56 GMT
>Organization: Dartmouth College, Hanover, NH
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>Hello:  We would appreciate any insight you folks might have into a
>problem we have recently run into with our site-directed mutagenesis
>efforts.  We've been using Promega's Alterd-Sites kit for several
>months to introduce single point mutations into several cDNAs with
>excellent results.  Lately however, we have had three instances
>involving three different mutational oligos and two different cDNA
>templates where we have obtained incorrect base changes at the site of
>mutation.  For example, in one case we wanted to mutate ...ctgAatt...
>to ...ctgTatt..., but instead obtained  ...ctgGatt... in 4 of the 5
>colonies that we picked and sequenced (the fifth colony had the
>wild-type sequence).  I thought that possibly there was an error in the
>sequence of the mutational oligo except that we got similar types of
>errors with two other oligos as noted.  No other base changes in the
>sequenced clones were observed.  Promega's tech support did not povide
>us with much insight into this problem.  Has anyone had a similar
>experience?  Do you know the explanation and is there a workaround so
>that we can come up with the correct mutations??  Any help would be
>much appreciated.

>Don St. Germain
Dear Don
I had the similar problems,too.  But I could not come out with any answer.

I-Jen Huang
GR8BN at cc.usu.edu



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