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qPCR primer troubleshooting please

qPCR primer troubleshooting please - PCR - Polymerase Chain Reaction Forum

qPCR primer troubleshooting please - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 01-17-2012, 12:35 PM
Pipette Filler
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Default qPCR primer troubleshooting please



Hi everyone,

So, my first post on this forum is related to troubleshooting, so I wonder if anyone could give me some suggestions.

I have re-ordered a pair of primers against gene X, which I have bought from a company in lyophilised 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 and right concentration of primers 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.

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 with this.

best wishes,
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