Hi,
I have been attempting to run a qPCR for a gene on some immortalised cell line mRNA. I have BLASTed the primer pair and the only result that appears is my gene of interest, no other transcript variants etc of the same gene or a different gene.
I have run the gradient PCR for the primer pair and used the appropriate annealing temperature, however, there was not much difference in any of the temperatures run and all of these gave a single band.
When I have run the qPCR, for most of the samples I am getting 2 melting peaks, around 5 degrees difference from each other. I have also run a gel of the qPCR products which have two or more bands on the gel.
The negative controls did not produce anything and I run a different gene on the same plate which gave the expected results/melt curve.
I have tried decreasing the acquisition times of each cycle from 30 seconds to 10 seconds, which is optimum for the machine that I am using, however, the result was the same.
Does anyone have any idea about what could be happening or how I can fix this?
Many thanks in advance.
- Hi William
- Are you using the same annealing temp in your qPCR as actual PCR?
- If so then what you might be seeing if the two bands are close is secondary structure lead to 2 different conformers
- The best way to determine that is to sequence them
- If the same amplicon then a technical artifact
- You could also test the conformation theory by trying to eradicate it
- Add 5% DMSO to your qPCR sybr green reaction and also before you load your qPCR products on an agarose gel denature @ 95C for 2 minutes, followed by 5 minutes on ice before running them on a 2% agarose gel
- How large are they by the way?
- If much more than 150bp you could also redesign your second primer to make amplicons ~ 50- 70bp (no smaller). That might help
Hello
If in the agarose gel you see a single band, but double peak in the melt curve, it means that maybe your agarose does not have enough resolution to visualize two bands with close fragment size. You can try a 2% or 2.5% gel with TBE buffer (Not TAE).
Consider another point; Its good to have same type of master mix for both PCR and qPCR reactions.
Sorry, can you rule out the idea of a possible contamination about the work solution of one of your primers William John Mason ?
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