Hi everyone, I am trying to insert a 24 bp fragment into my gene of interest using site directed mutagenesis via whole plasmid PCR. Its not working out, any suggestions about it? My mutagenic primers are complementary, 48 bp long and have Tm 69.7*C. I am using Phusion DNA Pol with the following cycling parameters.
1)95*C- 30sec
2)95*C- 30sec
3)55*C- 1min
4)68*C- 3min (my plasmid is 2.7kb)
go to number 2 18 times
5)68*C- 30sec
6)4*C- Hold