I am attempting to insert 6 Histidine codons and two restriction sites just
prior to the stop codon, and have not succeeded with site directed
mutagenesis methods. I keep getting deletion mutants. So I switched to
PCR to try to insert these 30 or so bp.
I'm looking for a 1kb product, and I am using a 5' primer that anneals
perfectly to the template and a 3' primer which anneals for 30 bp each at
the 5' and 3' ends of the primer, but also has a big loop of 30 bp in
between the annealing spots.
I think that the large and difficult 3' the primer is the reason that I get
no product. The template, dNTPs and Vent polymerase all work, I get
results with control primers. I have tried different polymerases (pfu) and
varied the Mg concentration with Vent from 2-10mM in 2 mM increments, tried
adding 5% and 10% DMSO, and 100ug/ml BSA, all in separte reactions. In all
of these reactions I used a low annealing temp of 45 degrees Celsius, given
that the lowest Tm of the two primers is about 60 degrees. Still, I see no
product under any of these conditions. The 5' primer with a control 3'
primer seems to yield product, so the culprit seems to be this enormous 3'
primer. (Not surprisingly.)
The only thing I can think of to try is to design a 3' primer which anneals
only at the 3'end of the primer and allows the His codons and restriction
sites to dangle.
Before I attempt this, are there any suggestions I should try?