How to design RT-PCR primers in-frame for expression vector? - (Mar/05/2012 )
I have a few sets of primers from a paper, for PCR amplifcation of cDNA. I want to use these primers for ligation into an expression vector for eventual expression. If the paper mentions that these primers could be used to create PCR product for expression, does that mean that they are already designed to be "in reading frame"? I have modified these primers to include restriction sites. How do I determine if my primers will amplify in the correct reading frame for eventual expression?
Thanks in advance!
The one thing you need for in-frame expression is to have the ATG start codon in place. If you are making primers for producing a full length protein, the easiest way to ensure that they are in frame is to include the ATG and the remaining first 15-18 bases of the sequence. The reverse primer should be the reverse complement of the last 20ish bases of the sequence.
You don't have to worry about the bases before the ATG (though you can add a kozak sequence to enhance transcription), as the promoter takes care of binding of the transcription factors.