is there a problem with running a PCR and then running the same system again, with the same primers, with the product of the first reaction to further amplify the product? I m using a nested PCR for detection of Theileria parva with Tp2 antigen, the first PCR has outer primers and the second one Inner Primers.
I m getting faint bands at exactly the expected size with the second PCR (nested). Does it make sens to repeat the second PCR with the same primers, using products from the nested PCR for improving the amplification? because the positives samples are going to be sequenced.
Hi Gaston, I would suggest don't use the same primers for second round of PCR. It does not make any sens to do this. Nested PCR intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites.
Hi Gaston, I agree with Lalan. The nested primer will increase the specificity. But in case your problem is just the sensitivity, you may also reamplify with the same primers. We do that once a while for cloning purposes when the first PCR results in a very faint band.
Hi Gaston. I agree with earlier experts' answers: don't run with the 2nd primer set once more. Did you include a negative sample in your assay? Is it in the gel shown? If it is empty, go on then and sequence it. Otherwise, repeat with negative control until it works like it should.
If you have to repeat this assay many times, you might consider using a different polymerisation enzyme. I also needed to do nested PCR with regular Taq, then I changed to Phusion High fidelity or hotstart enzyme. I got much better bands then and could do the same assay with only one primer pair (no nested needen anymore).
Good luck Gaston
Thank you for your answers to my problem,
Yes DUCO, I used two negative controls and there was empty in the Gel. I forgot to share the overall picture of all samples. but the negative was empty.
I m using a Bioneer Kit and then it is impossible for me to change the Taq.
Our sequencing unit is asking at least 25 ng/ul after purification, which is difficult to obtain from my amplification.
Dear Amzati,
How many amplification rounds does your 1st and 2nd pcr count?
It does not make sense to me. If the initial primers have failed to amplify well then why use them again. Using nested primers the effective genome size is now only a few hundred bases of amplimer so the nested primers will be highly specific and I would expect a cleaner and much better amplimer and a high yield. This,in itself, may be a problem so only a dozen cycles on a diluted template should be sufficient as too much amplification leads to amplimer concatenation and large size products
Thank you to you all for ths troubleshooting,
My PCR worked and I sent PCR product for sequencing.
Gaston AMZATI
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