Primer Design Urgent! - (Apr/21/2011 )
I have an expression vector whose MCS is limited to AhdI, PacI, FseI and EcoRV.
My goal is insert my gene of interest which is to be stored in a pcDNA the restriction sites of this vector is not identical to my expression vector. Therefore I must design primers to include restriction sites of the expression vector. It was suggested that I cut my cDNA piece out if the vector with a compatible enzyme (EcoRI) then purify the piece&PCR the cDNA from the gel with a adapter primer. How do I design this adapter primer? Will I be using the complementary restriction site (EcoRI) and then add the known restriction site of the expression vector.
Unless there is a specific problem, it is far simpler to PCR amplify the gene of interest from its original host vector. I recommend using Phusion polymerase from NEB to d i the job.
The desired restriction sites can be added to the 5' end of the primers. Donot forget to add at least 6bp of extra bp to flank the restriction site.