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I am creating a transcription template for expression in PURExpress and was confused about the annealing temperature to use.

I have two primers with the T7 promoter and an RBS on the forward primer and a termination sequence on the reverse primer. The melting temperature of these primers are 84.4 and 83.8 degrees C. The melting temperature of the regions that are complementary to the gene of interest I want to amplify by PCR are both about 65 degrees C.

What annealing temperature should I use a h? Since at the beginning of the PCR reaction I will have mostly template that will have the complementary sequences of the primers binding to the GOI and not the 5' and 3' extensions.

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It seems the answer, which may be context specific, is to use an annealing temperature about 5 degrees below the entire oligo melting temperature. I did not get any bands at lower temperatures.

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I have to add a comment that I asked PI who focus is synthetic biology, and that PI told me to use an annealing temperature 5 degrees below the melting temperature of the area of the primer that bind directly to the starting template. I think the answer is somewhere in the middle. –  Kevin Sep 11 '13 at 20:47
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