Tagging a gene with HA and FLAG using PCR - (Nov/21/2012 )
I´m trying to introduce HA and FLAG tags right after my gene(C-end). Since primers for this reaction will be around 60 bp I think i should do it in two steps. First add HA tag and in the other reaction add the FLAG tag. with the primers so how can they to be compatible with the template?Can anyone suggest...anything?
You shouldn't need to do it in two steps, I have used tailed primers of over 100 bp in length successfully.
Assuming that you are wanting to then clone the tagged gene your primers will be something like : 3-6bp RESITE 3bp ATG HATAG 3bp FLAGTAG 3-6bp GENEOFINTEREST (with or without the ATG on the gene of interest, approximately 20 bp of the gene). You can also add a consensus Kozak or Shine-Delgarno sequence upstream of the start codon to enhance expression of the gene in eukaryotic and bacterial cells respectively.