Problem with New qPCR primers - help! - (Jan/14/2012 )
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
Usually companies provide instructions for reconstituting primers at a strong concentration- have you diluted this further to your working concentration before using the primers for your PCR (sorry if that is stating the obvious, its just you haven't said in your original post if you did this or not...)
How long did you left the dryed primers in the water before aliquoting? I usually leave it overnight, shaking at RT till evening, and then the night in the fridge and then mix again and aliquot in the morning.
And for a better stability we use 10mM Tris pH8 to dissolve primers, but that should not have such effect.
That's alright, I don't mind at all elaborating as this is really puzzling me. The company specified a volume of water to add to attain 100 pmole/ul. I then made a 10uM working solution for PCR as recommended for the Phusion Pol kit. With the first batch of primers before I tried using them for qPCR (where they were used in a primer-probe mix at 750 nM and 900 nM for forward and reverse, respectively) but had no signal at all for the target gene.
As for how long I left them for, my supervisor hasn't mentioned this aspect of primer reconstitution to me. I just left them for a few minutes on the benchtop and then gently mixed them.... Ah, so if I went back to the stock solutions, thawed them and put them on a shaker overnight as you suggested I should get them both working?
Thank you both for replying so quickly and making these suggestions
Honestly I doubt its the reconstitution time, I rarely leave my primers for more than a couple of minutes before gently mixing and then taking an aliquot for dilution.
I dissolve in TE, vortex, and then aliquot. I think you are wasting your time doing much more.
Thanks everyone. My primers now work well. Possibly I might have just had a bad batch before. I've also started storing the primers in TE rather than water, so thanks for the suggestion, phage434 :-)