i have a problem to insert 39 bp into pET by using KOD plus mutagenesis Kit.
39 bp was attached to 5` of forward primer and i conducted preheating to this primer before using for PCR.
First experiment :
i used 7 cycles of PCR, 7 minutes annealing and elongation time, digestion duration was 3 hours, ligation duration was 2 hours, then after transformation into E.coli DH5alpha, i got no colony. There was no problem with transformation step because i used transformation control.
Second experiment :
I increased cycle number to 12 cycles. I got 3 colonies after 12 hours incubation but after checking the sequences it was wildtype, no insert sequences.
Third experiment :
I increased cycle number to 20 cycles and i got no colony.
I guest my insert sequences is too long but i did not get the reference of maximum bp to attach in 5` primer.
Does anyone has suggestion or experience with KOD plus mutagenesis kit to insert sequences?
thank you so much