epitope tagging of protein - (Sep/04/2014 )

Hi there,

I am trying design a strategy to epitope-tag my proteins of interest and I just want to make sure my strategy is okay.

N-terminus tagging:

forward primer: 5'-protective overhang-RE site-Kozak sequence(CAAC for Drosophila)-ATG-tag sequence-gene specific sequence(starting from the second codon)

reverse primer: 5'-protective overhang-RE site-gene specific sequence(including the native stop codon)

C-terminus tagging:

forward primer: 5'-protective overhang-RE site-gene specific sequence(including the native ATG)

reverse primer: 5'-protective overhang-RE site-stop codon-tag sequence-gene specific sequence(excluding the native stop codon)

I also have two question:

1. should the 5'UTR and 3'UTR be included? why or why not?

2. in the situation where the gene's native stop codon is not used, should i use two tandem stop codons to avoid reading through?

Thank you in advance for everyone's reply.

Looks fine. You might want to add a kozak to the forward primer for the C-terminal tagging. Don't forget to reverse complement the tag sequence for the C-terminal tag too.

1) No, think about the name UTR -that'll tell you why.

2)You can do that, ideally you should put stop codons in all potential reading frames.