Wednesday 4 May 2016

molecular biology - Primer design for introduction of restriction sites flanking a gene of interest

I think the best way is option #2:



Suppose that your gene of interest is AAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGGGGGGGGG



and you want to insert EcoRI restriction site GAATCC



Then your Fwd primer will be GAATCC AAAAAAAAAAAAAAAA



and your Rev primer will be GGATTC CCCCCCCCCCCCCCCC



But in general, it shouldn't matter which option you choose, as long as you calculate the Tm of only the part of the primer that hybridizes to your template.

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