I am trying to design a reverse primer with an affinity tag ( C-term affinity tag). Perhaps, my original protein sequence already has a stop codon at the C term. Could anyone suggest the correct way of designing a primer in such a case?
Design it so that you insert the affinity tag just before the stop codon.
When designing a reverse primer with an affinity tag for a protein that already has a stop codon at the C-terminus, there are a few options to consider:
It is important to consider the effect that the tag will have on the protein's function and stability, and to test different strategies. It is also important to check that the primer sequences do not create secondary structures or binding sites that would affect PCR efficiency or specificity.
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