Help: MORPH and Stratagene mutagenesis kits

Dr. Peter Gegenheimer pgegen at kuhub.cc.ukans.edu
Tue Jun 4 16:07:22 EST 1996


In <Pine.SOL.3.91.960604093029.12804B at muss.CIS.McMaster.CA>, u9310871 at MUSS.CIS.MCMASTER.CA ("A.S. Sohal") writes:
>Hi! My name is Avtar and I have been experimenting with some new 
>site-directed mutagenesis kits.  In particular, I have been working with 
>the MORPH single-primer site-directed mutagenesis kit and the Chameleon 
>site-directed mutagenesis kit from Stratagene.  I was wondering if anyone 
>has had any success with these kits.  I am having trouble isolating my 
>treated plasmid from the mut S cells after transformation.  The cells 
>grow in LB-amp so the transformation to gain amp resistance must have 
>been successful.  However, attempting to isolate the plasmid by means of 
>a alkaline lysis mini-prep has been unsuccessful in producing any DNA.  I 
>hope someone can save me.

This is probably not your particular problem, but the fact that a bacterium grows in the presence of AMP doesn't mean it has a plasmid ... not if its a gram-positive bug that fell off one's hands.
Also, you might try the Holmes&Quigly boiling miniprep, which is probably gentler on plasmids. Other posters should help out with the genetics & details of the mutagenesis itself.

P.Gegenheimer/U. Kansas Biochemistry/Lawrence KS 66045-2106



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