Epitope tagging - Several basic questions (Nov/20/2007 )
Hi. I am doing some epitope tagging by PCR for the first time and have a few trivial questions:
1) In primer design, I have to add two nucleotides between the restriction site and the start codon& tag to make it in frame. Does it matter what nucleotides I add? Are there any resulting codons that should be avoided?
2) After PCR product restriction, do I have to do a fragment gel purification, or can I run the restriction mixture straight through a Qiagen column? I mean, 2-3 nucleotides cleaved from each side probably will not be retained by the column, right?
Thanks for answering!
1) In primer design, I have to add two nucleotides between the restriction site and the start codon& tag to make it in frame. Does it matter what nucleotides I add? Are there any resulting codons that should be avoided?
2) After PCR product restriction, do I have to do a fragment gel purification, or can I run the restriction mixture straight through a Qiagen column? I mean, 2-3 nucleotides cleaved from each side probably will not be retained by the column, right?
Thanks for answering!
1. Don't add nucleotides that will result in stop codons.
2. I wouldn't do a gel purification unless you had multiple PCR products produced by the PCR reaction
Usually we add 2 glycine between tag and start codon of transgene. But make sure the addition of 2 bases is fine in terms of amino acids.
Also we run the PCR product on gel after digestion and purify it thru columns.