Hi all,
I'm attempting to do a single nucleotide mutation in a 10.1 kb plasmid. I've attempted to do this with various quickchange primer pairs with no success so I designed primers for the Q5 site directed mutagenesis protocol. My primers end in 2 GCs, have limited propensity to form secondary structures or dimers, with a Tm=66.3C. Most recently, I ran a 2 step PCR with Phusion polymerase and an extension time of 8 min. I ran reactions with 50 ng, 25 ng, and 10 ng of template as well as a reaction with 0.25 uM primers. Since Q5 is exponential, I ran the completed reactions on a gel and saw a band in each at about 1.5 kb. Since it was a 2 step, off target amplification seems less likely, but I'm not sure what else could be the problem. Any suggestions what could be the problem or what to try next?
Hi Xander,
I've had some problems with the Q5 mutagenesis kit before, I also didn't get a PCR product of the right molecular weight. What worked for me was to make my own Q5 PCR reaction mix, instead of using the mastermix that comes with the kit (you can get Q5 polymerase and the reaction buffer from NEB). I then performed a gradient PCR with different annealing temperatures to find the sweet spot, extracted the DNA from an agarose gel with a gel elution kit and continued with the KLD reaction, transformation etc. I'll link vou the protocol I used, maybe it helps you.
All the best, -Ben
Dear Xander
i'm never use the Q5 kit so i cannot suggest you solution specific for this kit.
In case you will not able to solve your issue, for single point mutagenesis of a plasmid you can use the PIPE cloning approach.
It do not require any specific kit, just a good high fidelity polimerase able to amplify so high DNA fragment (i tihnk you can try with Kapa Hifi of pfu ultra fusion II) and mach1 chemically ultracompetent cells from Thermo.
Attached you can find a paper that describe you the approach.
good luck
Manuele
There is a modified SDM method proved much more simpler and efficient https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91, might be worth trying it if you are still struggling to get your work done.
Perhaps try PCR-ligation-PCR mutagenesis described here Article PCR-ligation-PCR mutagenesis: A protocol for creating gene f...
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