Intensifying signal from positive control - (Jul/29/2013 )

hi All,

I have troubles with my PCR. I want to genotype transgenic animals. I have many troubles with it, like for first shot I got some positives, and with later repeats of PCR all my samples come out as negative, while my positive control stays nicely positive. And this is the thing: it is not enough, that my positive control stay positive only, but the signal from the positive control is increasing in later PCRs. Before I´m setting up the reaction, I usually make a dilution of ALL my samples to have the same concentration for each (like 100 or 75 ng/ul), and then I use 2-3 ul (150-300ng) for my PCR. I do the same with my positive control, so it should have the same concentration at all times. and still I experience an increase in signal (comparing, for example, to the signal of the DNA sizemarker, of which I also add always the same amount to the gel - so it is not the fluctuating level of EtBr, which I also try to add always at the same concentration). Does any of you experienced anything like that? I´m going crazy already with my PCRs :(

This, of course is not my main trouble, my main trouble is not getting positives :P, but it is also something annoying that I cannot explain.

How do your negative controls look?

Both my NTC (water instead of DNA) and negative (DNA from surely negative animals), and also all my other samples (sadly :P) are very-very negative. Only my positive control is positive, but it gets more and more positive with time. Any ideas what can it be? Can I still trust my PCR? Are really all my samples negatives, or something is not reliable, and there is hope for some hidden positives...?

This suggests to me that your PCR conditions are marginal -- sometimes they work, and sometimes they don't. You could try a gradient PCR, or titrating Mg++ concentration. You could try adding enhancers (my favorite is 3-6% of a 1M Betaine solution). What is your PCR volume? 2-3 ul of a 10 ul reaction is a lot, probably enough to make PCR inhibitors a factor.

My PCRs are 20 ul.  I repeated the PCR with different volumes of template, all stayed negative (while my positive control - which is also a genomic DNA - stays positive). The trouble is that I don´t really know to what should I titrate my reaction: my positive control works, no matter what, and my samples are all negative, and they stay negative every time. This phenomenon, that I have a positive signal, which is not there in later PCRs is actually something new. I always extraxt the DNA from the samples using the same method, and I am also working with several primerpairs paralell (usually with 2 indepentent pairs, in independent reactions), so it would be weird if the reaction would be that marginal in every setup but only for only for the new samples, when old positives are still quite reliable positive controls. And I still don´t get how gets my signal stronger with time. At this point I am mostly afraid of (1) false negatives, due to PCR inhibition, and (2) false positives, due to some kind of contamination. How can I be really sure that my PCR is reliable, if both can be an issue?

I'd suggest that you try diluting your positive control to establish the sensitivity of your assay. After doing so, you could try adding a barely positive amount of your positive control to the normal amount of your (expected positive, but negative) controls. This would tell you if you have inhibitors in the test samples.

Is there anything unusual about the sequence? Very low regions of GC over around 30 bp can require lower extension temperatures, for example.

With highly diluted positive template, you should more easily be able to optimize the reaction conditions (annealing, extension time, Mg++, additives).

Your controls will (in principle) tell you if your assay is working. Why are you certain your expected-positive controls are really positive?