WHOPS and site directed mutagenesis

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Mon Nov 5 00:57:12 EST 2001


Dear Michael, 

What is 1) WHOPS and b:-) Schmidheini HPLC pure quality (see web reference)?

I'd expect nicked circles, according to the nice images in the stratagene or 
invitrogen (those who sell this expensive sdm kit) catalog

Do I understand you correctly that you would like to introduce 2 different 
mutations at different, but almost adjacent postions at the same time?

Then you probably also might have your primers synthesized with 2 degenerate 
bases at the respective locations. I'd suspect that with your proposition, one 
primer will cure the mutation introduced by the other and slight differences 
in copy numbers introduced at the beginning might end up in one product in 
abundance.

Regards,

Wo

> Dear All,
>         I remember a tread about using two primers, plasmid DNA and DpnI
> to do site directed mutagenesis (actually this is described very nicely at
> http://www.ikp.unibe.ch/manual/site-dir.html).
>      Could I ask you all a question: do you think the PCR in WHOPS results in
> nicked circles or longer linear products (similar to rolling circle DNA
> replication?).  If it did then the two mutagenic primers could be separated and
> double the potential for introducing mutations.  Thanks for any comments. 
> Regards, Mike Witty.
> 
> 


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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.O.C.
e mail wolfsc at ibms dot sinica dot edu dot tw
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