QuikChange site-directed mutagenesis of 1 AA was performed for 16 cycles according to the manufacturer's instructions (Agilent Technologies).
Mutagenesis efficiency = 97%, transformation efficiency =99.8%, and there was an average of 1.5 colonies using the dnpI control. I got an average of 150 colonies, but when the sequences were sent for sanger sequencing... but the mutation is not present?
These were my primers:
F: 5'-ctcagatccggaggcgcgagcacccaccctat-3'
R: 5'-atagggtgggtgctcgcgcctccggatctgag-3'
Hi Scarlet,
It sounds to me like since you are getting a significant number of colonies even after Dpn1 digestion compared to your negative control, that there is probably an issue with generating the mutant sequence during the PCR cycles, which points to a possible issue with your primers. Without knowing the details of what mutation you are trying to make in which gene, I can't help you too much, but when I do site-directed mutagenesis, I make sure the mutation to be created is located in the middle of my primers and that there's 16-18 nucleotides either side of the new codon. I also ensure the primers are complementary to each other. If this is similar to what you've done, I might suggest adding a small amount of DMSO to your PCR reaction mixtures (3-5% total volume), or reducing your annealing temperature to something like 45 degrees C. Ensure you use a DNA polymerase without 3'-5' exonuclease activity (e.g. Taq), otherwise the mutations you generate during the PCR extension steps will be corrected to the original template sequence, therefore no mutations.
Best of luck!
Jonathan
Hi,
After many years of successful site-directed mutagenesis with the Stratagene QuikChange system, we started having a hard time (although we were not getting any colonies at all, or the few we'd get had no mutation).
We switched to NEB, which uses a different primer method, and we had much greater success; it's the "Q5® Site-Directed Mutagenesis Kit"
Good luck
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