For downstream fragment analysis (T-RFLP), I am running PCRs with primers labelled with the fluorescent tags HEX and 6-FAM. After having some trouble gaining enough product, I now do a primary PCR with the same primers but unlabelled, then a secondary PCR with the labelled primers. The amount of product may be sufficient, but possibly not. Do you have any advice or experience regarding the optimisation of such a PCR protocol?
Thanks Catarina, I will probably combine products from more than one PCR. I use 1ul of DNA template, but have tried T4 gene 32 protein to mop up some of the PCR inhibitors, with varying effect.
I have done a lot of PCRs on bacteria using a FAM labeled primer, and I haven't seen a huge difference between the unlabeled and labeled primers. I would consider adding something like BSA, changing your annealing temperature or magnesium concentration, or increasing the number of cycles. Or you could try purifying your DNA further if that is reasonable.
I would second Catarina's suggestion of pooling PCR products. I also used to perform T-RFLP analyses and combining products from 2-3 reactions during the PCR purification step solved any problems to do with yield.
The one thing that has made the most difference to the success of my PCRs was to change Taq DNA polymerase. I was using the cheap Fermentas recombinant Taq, but then happened to try a sample of Bioline's MyTaq HS Red mix (https://www.bioline.com/h_prod_detail.asp?itemid=272), and the rate of success, in addition to the brightness of the bands, are the best I have had so far with this particular root endosymbiont... It seems if the Taq is limiting the success of your PCR, it is very difficult to improve the yield!
Try contacting Matteo Girardi in our lab. He is a member of ResearchGate.
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