I run qPCR experiment by using sybr green assay. I run 10ul reaction. I get nice melt curves. How ever, the amplification curves are plain not picking up the threshold. Also my endpoint PCR shows good results.
hi,
The melt curve mainly indicates the melting temperature (Tm) of a target and the amplification can identify nonspecific PCR binding also. Multiple melt curve peaks tell about primer dimer. Here, in your case, the important thing is cDNA synthesis and the compatibility of the synthesized cDNA with SYBr green. The regular cDNA synthesis kit does not comprise polyA which is essential for such a small-size miRNA conversion into cDNA.
Regards
Saurabh
Please provide pictures on which all number and characters are legible.
Check the settings to make sure the camera is set up to measure amplification data. It's supposed to be the default, but folks can turn it off.
Run your reactions on an ordinary gel. You should see bands. Check that this is the case. If yes, something is wrong with the fluorescence acquisition. If no, something is wrong with your melting curves. But as Katie A S Burnette said, it‘s likely the former.
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