Site Directed Mutagenesis Problem - (Mar/03/2005 )
I am using Stratagene Quikchange SDM kit and protocol to mutate my gene of interest which is inserted into pET151D TOPO from invitrogen. I got the sequencing result and the gene is mutated. However, there is an extra 82bp in the middle of my gene near the mutation site. I checked the sequence of that 82bp and it resembles the primer sequences that I used. My primers are 29bp long. In that 82bp, i can see several segments that matches exactly with my primers. I am already using an annealing temperature of 62C. Anyone know how to solve this problem?
Thx
anyone?
Thx
HI Densin
I've had a similar problem where I see my primer sequence duplicated upstream of where it should be. I"m entirely sure why this happened, but I sequenced a second clone and that was okay.
Kerry
How many colonies have you checked, and if more than one (sequenced) do they all have the problem?
Seems like a primer dimer/hairpin problem to me. Do the primers form hairpins? If so, you might be lucky and fine a correct clone (you might be unlucky as well of course). Try to create a new primer with some silent mutations so that the hairpins in your primers no longer form might also be an option.
I got the problem solved I believe. Just to share what might be the problem. To vairus: Yup, all of them have the same problem.
So what happened is I did not mention in my first post that when I run 10 ul of the reaction on agarose gel, there is a smearish background in addition to the single band that corresponds to the size of my plasmid. I adjusted the percentage of DMSO I used in the reaction (from 5% to 2%; since my gene is high GC content and without DMSO it does not even get amplified in the mutagenesis reaction) and I was able to reduce the background. Upon sequencing the clones that i got, I do not see the repetitive primer sequence in my mutated clones as mentioned b4 anymore and all 5 of the clones that I sequenced are mutated with the correct mutation.
P.S. I posted the first post 2 years ago lol 
So what happened is I did not mention in my first post that when I run 10 ul of the reaction on agarose gel, there is a smearish background in addition to the single band that corresponds to the size of my plasmid. I adjusted the percentage of DMSO I used in the reaction (from 5% to 2%; since my gene is high GC content and without DMSO it does not even get amplified in the mutagenesis reaction) and I was able to reduce the background. Upon sequencing the clones that i got, I do not see the repetitive primer sequence in my mutated clones as mentioned b4 anymore and all 5 of the clones that I sequenced are mutated with the correct mutation.
P.S. I posted the first post 2 years ago lol
Hi
I am performing site directed mutatagenesis to my gene of interest; the first two PCR reaction with the right mutations were fine.
but when I wanted to perform the same PCR for different mutation (or the same pervious mutation) I repeated to confirm., Nothing seem to work at all. sometimes I get wrong mutations, sometimes i get onlyt the wild type template, other times I get nothing at all. I have changed everything, tried all over again, tried different machine, nothing seem to be working. I only have one month left to finish my thesis, but nothing seem to work. ANY HELP would be so much appreciated.
Thanks