When I first started doing the PCR, I did not observe bands for my no template control (NTC). However, from some point onwards, I have been seeing bands for my NTC. I have 5 pairs of primers and 2 of them gave quite a few bands while the other 3 gave bands at 100bp region (100bp is too big for primer dimer right?). So I changed everything, from reagents to water and I re-diluted my primers from the stock (I assume the stock is fine as the first few rounds of PCR had no issue). But the issue still persists. I then tried the same set of reagents on a separate primer pair that previously was used before and had no NTC issue. It was run together with the 5 pairs of primers that had issue using same reagents. The separate stand alone primer pair had no NTC issue while the 5 pairs in question still had the same issue, even though they were prepared and run together. I tried adding DMSO since it could be due to secondary structures of the primers but it did not help as well. Much appreciated if anyone knows what is wrong with the set up. Thank you.
In my experience, 100 bp is too large for a primer dimer. Did you use PCR grade ultrapure water? Are you sure it is pure? You wrote that you didn't see any bands at first. It's possible that the primers/water were subsequently contaminated and I think you used the same water to re-dilute the primers from the stock. Try changing the ultra pure water. What about your samples? Did you get the right band or not?
Hi Monica Auteri thanks for your answer. I have used molecular grade nuclease free water from Qiagen and I have used new box of the water when I re-dilute the primers so I did not use the same water twice. Basically, when I was troubleshooting and repeating, I did not use the same reagents as previous. For my samples, for some of the primer pairs, I get the right size band together with non-specific bands while the rest are all non-specific bands. At first I thought is because the PCR conditions are not optimized but with the NTC issue, it may be caused by the same cause that created the bands for NTC as for those non-specific bands I seen for the samples.
In some cases it is really difficult to know what happened during the PCR. From your answer I understand that you don't have the same problem with different pairs of primers (and using the same reagents). Probably for the same unknown reason the primers are contaminated (maybe contaminated filter tips?). Are you running your PCR under a laminar flow cabinet?)
Other than that, the only thing you can try is to increase the annealing temperature, in order to try to avoid this non-specific bands.
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