PCR Primer Design with restriction sites - (Dec/21/2004 )
i designed a primer for a promotor region of fcerg1 gene to study its snps effects via reporter gene study.
i did a epcr of the primers from ncbi and it matched perfectly to the corresponding locus, but after i added a restriction sites to the forward and reverse primer mul I and Xho I respecitveöy for PCR cloning in my PGL2 basic vector, the primers dont score a epcr hit.
is this a serious problem.... will it affect the actual amplication of my product during real PCR.
please explain me how to proceed?
No, it won't. Because you have added some bases to the 5' end of both primers, the program can no longer find a match for the new sequences. So just go ahead with your PCR.