deletion PCR? - I need help (Aug/31/2007 )
I need to delete a sequence in my plasmid by PCR. I found this method here: http://www.methods.info/Methods/Mutagenesi...R_splicing.html
but I think that method only applies to linear DNA, right? how can I delete a sequence in plasmid?
well you may digest your fragment in the part you want to delete and do the pcr on that template
alternative : in 2 tubes, divide your reaction in 2. I mean do 10 cycles with only one primer in each tube. Then mix the 2 tubes, homogenize well, resplit in 2 (a 50µl reaction is ok, seems that sames concentrations in 100µl are pretty bad)
That will allow to have single stranded DNA which can serve as template for PCR of the vector.