USS DNA and Mutagenesis

dnicker at dnicker at
Fri Jun 4 06:47:49 EST 1993

	Could anyone supply me with a tried and true protocol for the
preparation of uracil-containing SSDNA to be used in oligonucleotide mediated
site specific mutagenesis?  We are using Bio-Rad's mutagene kit, and I have
been having limited success (very low efficiency) so far.  
	Also, couls anyone explain the following observation to me-  In my last
tranformation of an attempted mutagenesis, plaques were observed (albeit a
small number) on plates containing transformed negative controls.  The controls
included primer 2. no ligase  (in the mutagenesis reaction).  I would have
not expected to see plaques on the reaction without primer especially.
	The net has solved my sequencing difficulties, now perhaps you can help
me with the mutagenesis??  E-mail preferred but I do read this group regularly. 
Sorry for the basic nature oif this question but as an Inorganic Chemist
trying my hand at these techniques for the first time I could use a bit of
adcdcvice.  Thank you in advance for any assistance.

							Darren Nickerson
							Univ. of Oxford

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