Perplexing result with site-directed mutagensis

Duncan Clark Duncan at
Thu Nov 24 06:46:20 EST 1994

In article: <3ajtdg$5jf at>  Donald.L.StGermain at (Donald L. StGermain) writes:
> 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.

This may be daft but it has been known for phosphoramidites to be labelled 
incorrectly by the manufacturer or for the wrong one to be put on a 
DNA synthesizer hence incorrect oligo. Alternatively could your 
mutation in some way be lethal and the E.coli is making it less 

My mind's made up. Don't confuse me with the facts!

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