Custom Primers - (Jul/11/2005 )
I have a very basic doubt in primer design. My interest is to design primers for target gene amplification from genomic DNA. The PCR product will be ligated into expression vector to be used for mammlian expression.
In this case, while designing primers, Unique RE sites are to be incorporated. My doubt is whether RE sites in forward and reverse primers should be different or same.
I have come across references where two different RE sites or similar RE site in both the primers are used. I am confused, please help.
thanks in advance for suggestions.
A few things....If you want to get expression it would be better to clone the cDNA rather than genomic-cut out the introns and just have coding sequence. If you include the introns the gene is likely to be too big to PCR anyway.
The two primers should have different RE sites which will allow you to clone the PCR fragment in the correct direction for expression. Also by having icompatible ends you won't get as much plasmid reannealing and will make the ligation much easier.
Thanks for your valuable reply.
Gene of interest is an intronless gene.