I am working with pET 21 B plasmid. It has polyhistidine tag. I am designing a primer and I don't want polyhistidine tag to be expressed. How can I prevent that? I hope I am clear. Thanks for your assistance.
Stop codon of your gene will prevent the polyhistidine tag to express, but a read through is possible. A stop codon (TAA is preferred because it is less prone to read-through than TAG and TGA) should be included in the reverse primer when no C-terminal tag is used. To increase the efficiency of termination it is possible to use 2 or 3 stop codons in series, as stop codon does not encode an amino acid.
Also, if you don't want the vector encoded T7 tag or its trace, use NdeI and any other restriction enzyme I.e. BamHI to XhoI can be used.
Thanks a lot. So, should I put the stop codon on both forward and reverse primers? Should I put the stop codon in front of the restriction cut site? I am using both NdeI and XhoI and the plasmid is pET 21B which has polyhistidine tag gene being encoded.