I'm having an issue with non specific binding of my primers. I've tried many primers and this is the least messy primer pair but I am still getting a second melt curve peak when doing serial dilutions on the qPCR. The peak is present in a 1:4 dilution at a much lower height and gone at a 1:16 dilution. Additionally, the peak at 82.29 C is higher than the correct peak at 77 C which contradicts my gel (lane 4 after the ladder). This peak is also present at the same concentration in another strain.
Because I am working with bacillus subtilis this cannot be an intron issue and it also cannot be a genomic DNA contamination issue since I have performed (-) reverse transcriptase controls (lanes 7-12 after the ladder) and while there is gDNA, the primers are specific in those controls. Everything has been done in parallel so any contamination should be present ubiquitously.
So I have two questions,
1) what might be responsible other than gDNA for off-target binding in a prokaryotic organism?
2) why would the qPCR machine indicate such a high melt curve peak for a product that according to my gel, is barely there?
The gel isn't run for too long, the correct amplicon is 127bp which corresponds to what I'm seeing in all my lanes and the 83 C melt curve peak corresponds to that larger product I'm seeing in my gel.
Hi Denise, If I see the same gel picture / melt results I always suppose unspecific products. I agree with Arthur, may be you unspecific band is go out of the gel. But in my experience some times I received single band and two or more melting peaks because of more sensitive detection using SYBR Green.
So, please try to add less Mg2+ in reaction, at least in 2 times. If it is not affect the reaction results, you also should add less primers (-30-50%). In the case of a good primers your unspecific bands/peaks should go out.
I hope my answer will help you.
Denise,
Arthur is right, your should switch to probe-based qPCRs.
regards
Alex
Hello Denise, I agree with Andrei and Artur. Try to check the quality of extracted DNA and make serial dilution of DNA to get optimal conditions.
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