Mutagenesis Problems

[heidi moss]

Dear Karen: 

		I experienced similar problems with the kit: I had 2 
mutant oligos (each with a 5bp mutation flanked on each side 
by 10 matched bp) + SalI selection mutant oligo that I had 
to anneal  to a 6kb vector. My first 2 attempts were 
unsuccessful. However, after the following modifications, I 
can happily say, my mutants are now ready to go:

	1) INCREASED phosphorylation reaction time to ON (our PNK   			
    was old, so I also ordered new enzyme and buffer)
	2) 400M excess of EACH oligo
	3) ***DENATURATION: this step is CRITICAL and I was 
surprised to read that Clontech only had a 3' denaturation 
step. I boiled mine for 10' to increase the probability of 
efficient denaturation.
	4) Ligase/Polymerase reaction: ON and at RT
	5) ETOH precip/concentration of DNA before each 
transformation.


I hope this helps!!
--Heidi