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
