insertion 39 bp with KOD plus mutagenesis Kit

[send_dy]

Hello guys,
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

[jlf]

1) What are the Tm of your primers, excluding the insertion part? Annealing temperature should be determined only by the sequence overlapping the template, excluding the insertion part. Add an annealing step adjusted to the annealing temperature of your primers.

2) What's your primer concentration? I find that full plasmid PCR works better using less primer. 0.2uM usually works fine.

3) How much template are you using? Too much template will lead to too many template-product hybrids, which will be digested by DpnI as well. Did you check if you got any PCR products? This method seems to amplify exponentially (unlike linear amplification of Quikchange) so I guess you should be able not need as much template

I don't think the insertion length should be a huge concern but I may be wrong.