I thought I would try asking on these forums once again for assistance as you were quite helpful before.
I have re-ordered a pair of primers against gene X, which I have bought from a company in lylophilised form. When they arrived, I followed the instructions to dissolve the primers in the indicated volume of RNase-free water. Then I gently pipetted the solution to ensure none of the white strands of primer were visible before aliquoting. I then did a standard PCR (ensuring I altered the temperature for annealing appropriate for my primers using the NEB tool and had enough cDNA for the reaction) against an old set of primers that are the same sequence as these new ones. Then I ran out the products on a 1.5 % gel to see if there is a product given in each.....
However, I have done this with two pairs of primers ordered against gene X now and both times I have not got any amplification, whilest I see a product of the expected size in the old primer reaction. The sequences are fine and identical in both the new and old set and I have used the ABI guidelines for designing real time primers in conjunction with Primer3.
Could anyone suggest where I might be going wrong here? I'm relatively new to using primers ordered in (before I have been given aliquots of each F and R primer solution to use), so if anyone can tell me where I'm going wrong it would be great if you could point it out, because this is beginning to drive me mad that I cannot get these primers working!!!
Thank you and I hope someone can help me out with this.
Tawny Owl x
Edited by Tawny Owl, 14 January 2012 - 07:43 AM.