I am using the T7 promoter primer or terminator primer with a gene specific primer to test my ligation before transformation, but the the Tm value of T7 primers is about 10C lower than my gene specific primer. I don't know what annealing temperature I should use in order to get a good band.
Baharak Mahyad
Dear Hao
Use 45C as your annealing temperature. I think in this temperature you will get a good band because both T7 primers and your gene specific primers would work properly but I'm afraid you would get nonspecific bands too. to discard these kind of band, after getting a proper band increase your annealing tm gradually until you get rid of nonspecific bands or you can purify your band.
good luck.
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