Hi,I have actually being trying to do almost this exact same...

J
Jimmy
Aug 17, 2011
protocol Protocol: PCR-RFLP Genotyping of Point Mutations in Caenorhabditis elegans
Hi,

I have actually being trying to do almost this exact same thing, only I have had no luck introducing a point mutant at the 3' ends of my primers in order to complete a restriction site (in the mutant version only). I believe its because I'm using a proofreading enzyme which
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Peichuan Zhang Answered Aug 18, 2011

Calico Life Sciences

Hi Jimmy,

I used very typical recombinant Taq (e.g., Invitrogen 10342-020) and it worked pretty well in my hand (home-made Taq also worked). I would recommend you not to use Taq w/ high proof-reading capacity.

The other thing that I have in mind is that you probably would like to re-design your primers. Put GC clamp at 5' end, but not more than 4 GC at the 3'-end. Treat them as primers for qPCR.

I run the gel for rather a long time (put a little bit of EtBr in the buffer), with the aim to separate the two bands, which differ just in ~30-bp or so (I don't really bother to check the 30-bp band though). In my case, I can always check the phenotypic readout for daf-2 mutations, which is dauer formation. If you have concern about the genotyping, you should also sequence the PCR product and check certain phenotype that are associated with the mutation.

Best luck,
Peichuan
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