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I am having a problem with the amplification curves in my Quantitative PCR having a very strange shape (please see attached figure).
The problem is sample specific, and is not due to the primers (on the same plate, both sets of primers worked fine in "Sample 2", and both failed in "Sample 1"). I'm just showing a subset of the samples here. About half the samples on this plate failed. I set up the reaction twice, and the same samples failed the second time as well, so I really do think it's a sample problem. The samples that failed were all from the same prep - I am thinking it's some kind of contaminant that was introduced during the prep (either in the RNA or cDNA step), but I'm not sure how to figure out what it is or how to avoid the problem in the future. Any ideas would be greatly appreciated!!
some other notes:
1. These end products of the QPCR ALL look fine (right size, proper band strength) on a gel. So it is not as if the weird ones are not amplifying.
2. I ran this on the StepPlus from Applied Biosystems, and the SYBR reagent I used was from BioRad. These are the same reagents I and everyone else in the lab has used successfully in the past.
3. The melt curves look the same for the samples whether they have "weird" or "normal" amplification curves
4. When I repeated the run, I changed the location in the plate each sample was in, so that in case it was a problem with the machine reading particular parts of the plate it should affect different samples, but the same samples were affected.
5. The samples are all fly cDNA made from RNA using Invitrogen M-MLV and oligo-dTs. I use a 2-step protocol where I purify the RNA (Trizol) then make cDNA as a second step.
