The first few qPCR runs the primers worked and I was getting signals in my samples/positive controls. However, now when I run another qPCR with the same primers I get no signal. I've tried using a fresh primer aliquot but that hasn't worked. The primer stock has been defrosted several times and is less than a year old so I'm not if its degraded or not. If it's degraded, would it make sense to slightly increase the primer concentration in my PCR reactions to get a signal?
Many thanks!
Primers can degrade from repeated freezing & thawing. No, just increasing the concentration will not be a proper fix. Just throw them away and order new.
Your qPCR reaction mix can also degrade from freezing & thawing. Most folks aliquot out "single experiment aliquots" to avoid this issue.
Good luck!
Hi Ali,
If you are using new samples, maybe the problem is not in the primers but in your samples themselves. If this is the case, add a sample which you are sure is positive for your gene of interest. Another possible cause is the master mix.
So Can you elaborate some details about your samples and how much primer did you use?
Best...
What is your primer concentration? Usually, I have no issues and primers can be thawed many times. As said above, it could be master mix, especially if you have thawed that many times. What is your cDNA concentration?
Often primers stored in water can absorb CO2 and depurinate or the water can have dnase activyty. If you add more primer and the pcr works with the larger amount of primer then degradation may have occurred.Order new primers and dissolve in TE....the tris buffers alkaline and the EDTA removes MG and divalent ions so inactivates dnase. Aliqiot the dissolved primer to minimise freeze thawing
Hellow Ali M Al-Rashed
Since your primers have thawed and frozen several times, it's more likely to be why your qPCR is not working. They reduce the efficiency and effectiveness of the qPCR. I would recommend purchasing new primers and preparing multiple aliquots. This will reduce repeated thawing and also avoid primer contamination. And as previously mentioned in a prior answer, "TE buffer" is preferred in long-term storage.
Best wishes!
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