In article <socenean-021195152917 at mac026029.ox.icnet.uk>,
socenean at icrf.icnet.uk (Loredana Soceneantu) wrote:
>> 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.
I belive the problem you are having is because of your choice of PCR enzymes.
You have used all the proofreading ones and these enzymes are very good at
chopping off the missmatched ends where your 6 His etc are to be found.
Try again with good old Taq polymerase and this should give product. It
isn't usually neccessary to nail down the 5' end of the flanking primer,
but since you have already paid for the extra 30 bases, you may as well
use it. If you still have problems with the Taq pol you may need to order
a new 60mer and just have the 3' complementarity and have the extra bases
PS this is a 90mer? that is a huge piece of DNA for std synthesis, its
possible that by gel purifying ( end label and run on 10-15% seq gel) you
can lose the accompanying ladder of failed extensions and up your yield in
You may want to try a series of anneal temps as well.
PPS beautiful name
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu