6 replies to this topic

[Curtis]

The attached file is the melting curve of my qPCR. The purple curves belong to housekeeping gene. The other colors are my samples, most of them peak at 75C, same temp known for primer-dimer.

 

Now, my housekeeping gene Ct value is at 15-16 cycle (not shown here), but the samples rise at 26-28 cycle. I thought primer-dimer usually rises after 30 cycles. So what are these peaks? Could this be because of low concentration of template?

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[jerryshelly1]

I don't think you showing the melting curve for a primer dimer. If you look at each sample individually do you see a little peak near the left of your plot?

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[Curtis]

so is it a product? 

[jerryshelly1]

Are the peaks at 76C and 80C from the same primer set?

[Curtis]

no, as I said above, the housekeeping gene is purple, and the samples are green. thank you.

[jerryshelly1]

Ok... The decrease in fluorescence can probably be attributed to a lesser concentration of product. What do your Ct values say? A primer dimer melting curve would be shifted to the left. Why would a 15bp product melt at a temperature that is equal to a 100bp product? Just run it on a gel and see what size your amplicon is. If it is the predicted size...

[Curtis]

thanks, we actually did. we see a band about 100 bp. it must be nonspecific product because our pcr product is supposed to be 150 bp. so it is probably not primer dimer. so are you saying that primer dimer usually appears less than 75C? at about what temp exactly?