I am trying to introduce a 3bp change in a gene of interest and I am using the Quik Site-II Directed Mutagenesis kit to do that. I have been having problems as I do not seem to get any colonies not only on my sample plates but also on my control. I have done the following troubleshooting: my reagents have been checked and work, thermocycler has been calibrated, I ran a gel after the PCR but I do not seem to get any visible product but I have read that this might be due to the fact that there is very little mutated DNA; I have used XL-1 Blue as well as XL-10 Gold bacteria; I have tried transforming various amounts of DNA. After all these, I still cannot get any colonies at all. Does anyone have any suggestions why that might be? Any help is appreciated! Thank you!
Dear Veselina,
If you don't have visible pcr product (which is pretty common), I would try skipping DpnI treatment. If you get colonies, then probably your mutagenesis primers are not working...or something else during pcr. If you are sure your buffers and polymerase are working, I would check the quality of your primer, which should be at least desalted...HPLC would be optimal.
Good luck.
Dear Pablo,
Thank you for your response. My primers are desalted and HPLC purified so I do not think that is a problem. I will definitely try omitting the DpnI step and see whether I get any colonies, but all that would tell me is that something is not working before that I guess.
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