Hello dear colleagues. Im doing qPCR research of mitochondrial copy number in DNA samples from blood. Im using StepOne plus thermocycler and Applied biosystems 0.1 mL 96-well plates.
While doing standard curve analisys I noticed some strange curves that don’t match other replicates. I tried 2 StepOne plus thermocyclers and 2 bathes of plates and different DNA samples and reagents.
These are images of 3 runs with same template and reagents but 2 thermocyclers and different batch of plates. The same wells look strange every run. Even if i fill all 96 wells with replicates, there are allways many same wells that are lower than others, no matter what primers (mitochondrial or nuclear) i use. 1 thing that i noticed - in 1 well with such low curve there were some droplets on the wall after run, but not before and only in 1.
My PCR conditions are:
95C at 6 min
95C at 15s
60C at 10s
72C at 10s
What could be the reason?
Dear Sir
this can happen. It’s sounds very much like well contamination, e.g. dust or grease. I would therefore do one of 2,things
I agree with Laurence: well contamination is possible
But may be it is the result a heteroplasmy of mitochondrial dna??
Did you check melting curves for these extra products?
Thank you for your answers, but the problem remains when i use 2 different cyclers and 2 fresh plates from different batches, so it cant be contamination. And strange thing is that wells with low curves are allways the same ones.
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