I am doing alanine scanning mutagenesis using Pfu Ultra turbo DNA polymerase and XL1 blue supercompetent cells. My primers have the 3' overhangs. The annealing temperature I have used is 3C lower than the Tm of my primers. Upon DpnI digestion, I'm able to see the plasmid on the gel. But upon transformation, I get very few colonies and all the ones I have picked up and screened for are wild types. Can anyone help to increase my chances of getting point mutations? Previously, I have used the same protocol and got a lot of mutants. This time it isn't working. Thanks
Dpn I is not very efficient in the PCR buffer, hence the background colonies.
Try diluting your PCR reactions with 1X DPN I buffer (15 ul of 1x DPN buffer + 50 ul
of PCR +1.5 ul Dpn I works for me)
I dont understand what you mean by 3' overhangs on primers. The 3' portion of the primer has to perfectly align with DNA sequence you are amplifying (at least 12-15 bases on the 3' end).
In most cases, low efficiency of site-directed mutagenesis can be avoided by changing the primer design. Explanation and an improved designing method is described in http://www.biomedcentral.com/1472-6750/8/91. Go through the paper to see if the modified method can help.
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