U.S.E. mutagenesis

Tim Kirksey tkirksey at med.unc.edu
Thu Aug 22 17:12:54 EST 1996


Jane.Wyatt.1 at massey.ac.nz wrote:
> 
> Hi guys,
> I am about to try using the Unique Site Elimination (USE) kit from
> Pharmacia Biotech, and I am just wondering if there is anyone out there
> who has used it. I will carrying out the mutagenesis with my cDNA
> inserted into the pGEX vector ready for E. coli expression.
> Any advice or warnings would be gratefuuly received... I'm sure it won't
> be as easy as the protocol suggests!
> 
> Thanks heaps,
> 
> Jane.
> 
> E-mail: Jane.Wyatt.1 at uni.massey.ac.nz


Jane,
	I have not used the USE kit from Pharmacia, but I have used the
Clontech Transformer kit, which I think is based on the same principle
(i.e. using a second mutagenic oligo to remove a unique restriction
site-I think there are several kits on the market based opon this method
which was originally published by Deng and Nickoloff).  This method
worked very well for me and often gave mutagenic efficiencies above 80%.
However, it is important to choose a unique restriction site that is cut
very efficiently by thr restr. enzyme, since the completeness of the
digestion will affect your mutant yield.  I read somewhere that enzymes
that cleave the recognition sequences XGATCX (e.g. Pvu I) work poorly.
Good luck.
Tim (tkirksey at med.unc.edu)



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