All my ARMS-PCR primers have TM of 66.6°C. I am getting only the mutant allele and outer primers' band at 69°C. But the sample is homozygous for the wild type allele.
What result do you get from your no template control....you may have post pcr contamination of a reagent so that will always amplify.
ARMS relies on the mismatch making it impossible for the other primer to anneal at the annealing temperature. If you have the annealing temperature set too low then the wrong primer may be annealing to the mismatched sequence and giving an unexpected positive result. You may need to run a gradient of annealing temperatures with both primer sets on both normal and mutant dna to determine the best annealing temperatures but check the no template controls first.
Thanks @Paul Rutland! I will try this and let you know the result. One more thing, I have to use DMSO, otherwise get only nonspecific bands. I have done pcr with the outer primer set and used this pcr product as template and obtained wild type allele for that sample.
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