Inserting a protein tag via looped primer mutagenesis

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I want to add a five-residue tag to my protein without fussing around with subcloning and I figured that mutagenesis would be simpler (Edit: the protein is already cloned into a vector I just want to add another tag to it). I'm familiar with using site directed mutagenesis for substitutions but I've never needed to do anything like this. Is there anything special I need to keep in mind about designing my primers? I assumed it would just look something like:

25bp homologous sequence -- 15bp insertion for the tag -- 25bp homologous sequence

Am I way overthinking this?

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dazosan

Postdoc | Biochemistry

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Posted: 9 years ago
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