I designed SSR markers using MISA tool from the EST database downloaded from NCBI. The product size of one of the markers which I amplified in the PCR shows no specific binding. I also did gradient PCR but the problem persists. Can anybody help me?
Alex Ignatov
This is a usual problem if you selected primers without complete genome data. You can try primers extended for 2-3 base at 3'-end, or design one new primer - less frequent in the studied genome. Sometimes, new PCR kit can solve the problem. Try all DNA polymerase types that you can find. We tested up to 5 different types for SSR, and in some cases only one gave good result.
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