Hello, I have just started with some qRT-PCR for my reasearch and Im doing a calibration of my reactions. With all the primers I checked(5 genes) I get an amplification of my NTCs and NRTs, in a higher Ct than my standart samples. I suspected that there is contamination of the water or the SYBR green reagent, but the melting analysis of the PCR shows that the negative controls have lower Tm than the sample amplification Tm. as much as I understand, it means that Im dealing with primer-dimers. I get only one peak in my samples melting curve. my working primers concentraion is 400 uM. Should I lower this concentraion in order to eliminate primer-dimer formation? and if I cant eliminate this, can I still trust my results regarding the fact that there is no primer dimer formation in my samples?
Thanks!!!
If your primer dimers appear within a similar cycle range to your actual product, then this is a problem, and one probably best addressed by redesigning primers rather than lowering the concentration. In a pinch you could try increasing your annealing temperature to raise the stringency, though this may affect your reaction efficiency (so is not ideal).
If they don't appear until significantly later (say, 8+ cycles later than your product), then honestly? Don't worry about it. All your relevant measurements will have been made before primer dimer signal appeared.
Due to the exponential nature of PCR, seeing primer dimers is incredibly common: anything that CAN amplify, no matter how inefficiently, will amplify...eventually. All that matters is how fast they appear.
If your primer dimers appear within a similar cycle range to your actual product, then this is a problem, and one probably best addressed by redesigning primers rather than lowering the concentration. In a pinch you could try increasing your annealing temperature to raise the stringency, though this may affect your reaction efficiency (so is not ideal).
If they don't appear until significantly later (say, 8+ cycles later than your product), then honestly? Don't worry about it. All your relevant measurements will have been made before primer dimer signal appeared.
Due to the exponential nature of PCR, seeing primer dimers is incredibly common: anything that CAN amplify, no matter how inefficiently, will amplify...eventually. All that matters is how fast they appear.
When you have a lower TV in the negative control this is probably due to dimers, you can double check by running those samples on an agarose gel. In case you are definitely sure there are no dimers in your samples, but only in the negative control, you still can use these primers as it is.
Hello, I agree with John that if you have primers that tend to form dimers then the best way to solve the problem is by redesigning your primers. However, I would suggest the following tests to solve the problem:
1. Test you primer pairs using one of primer designing software to see if your primers form hetero- or homo-dimers and check the delta G value. If delta G is < -8 kcal/mole then it is better to redesign your primers because this means that your primers are annealing with themselves and this will make them less available to anneal to the DNA template.
2. If your primer pairs form hetero- or homo-dimers with delta G at the gray zone “-5 to -8 kcal/mole”, then you can try the touchdown PCR protocol. Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced. This usually improves primers’ specificity and gives them little chance to play with each other (make dimers).
Hello. I agree with prior speakers. You have fundamental empirical problems and you are approaching the problem ffrom a very esoteric and theoretical stand point
Redesign primers byt do things like screen for primer dimer formation; self annealing etc
There are many programs that will allow you to do that
If it helps find attached a word document I give to students when I teach them sound primer design: Apart from optimal intrinsic base content you will find links in this document to primer design programs that amongst other things will screen for primer dimers
Best of luck and let me know if I can be of further assistance
If you want to just let a program do it for you (i.e. be lazy), I find this site to be enormously useful for qPCR primers:
http://primer3.ut.ee/
Well Primer3 designed primers will also end up doing dimers at some point. After 35 cycles, what you amplify is generally meaningless. If dimers appear earlier, don't use the primers. If 1) your samples are amplified within your standard curve, 2) you don't have dimers in your samples, 3) the standard curve has good linearity and 4) you don't see dimers in the less concentrated points of the standard curve, then your results are OK.
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