I want to amplify 3.4 kb gene fragment with primers having the restriction sites of enymes which i will use further. before adding the restriction sites Tm of my forward primers is 54.4 and after addition of RE sites it become 63.4 , and same for reverse primer before adding Re sites it was 57.8 and after adding 67. now which Tm should i use in order to amplify my gene and also adding the re sites in it? Kindly guide me which reaction set up would be more friutful?
You should use the lower Tm, i.e. without the restriction sites, since these don't belong to the sequence you wish to amplify. Theoretically, you could increase the tmperature of hybridization after a few cycles, since the amplicons will now contain the sequence of the restriction sites, but there is no need to bother about that.
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