Hi,
I have been trying to optimize some new primers, but I am not sure what else to do.
My project consist in knocking down a gene infecting primary transgenic cells with cre adenovirus.
I designed the primers and using 4*(G/C) + 2*(A/T) I got 62C for both primers and fragment length of 157 bp.
I use Luna Universal Master Mix from NEB which they suggest: Initial denaturation at 95C for 1min for 1 cycle. Denaturation at 95C for 15s and extensions at 60C for 30s for 40 cycles and melt curve at 95C for 1s, 60C for 20s and then 95C for 1s for 1 cycle.
My qPCR results do not show a peak when analyzing the melt curve, but it shows amplification curves and the thermofisher software analysis provides a CT value and it concludes that it amplified.
To verify it, an agarose gel was prepared and it showed bands corresponding to the expected length and reduced on the treated group. This confirms the CT values I am getting on qPCR.
I have tried checking different ranges for the extension temperature: at 60C, 62C and 64C. However, I get the same wave patterns.
I am not sure what else to try or if the result of the melt curve is relevant.
Does anyone have had melting waves that do not correspond to the amplification waves and gel?
PS: I am using HPRT as endogenous control, the samples were freshly made and these primers are completely new, I just received them. I am also attaching the plots and gel for 60C.
Thank you
This is in fact a strange result. The correct sequence obviousely got amplified, and one would expect a clear melting peak. If the sequence extraordinarily GC rich?
However, as you show in a gel that the amplicifation is correct and specific, you actually don't need the melt curves. The data seem to be ok for a quantification.
Thank you for your answer @Jochen Wilhelm!
The GC content is 63% for both forward and reverse, with a total of 19 bp per primer. Forward with a self-complementarity of 6 and Reverse, 5.
Hmm, those are odd results! The melt-curve is the least informative part of a qPCR reaction so I'd believe your gel data more than the melt curve data. Double check your machine settings to make sure it was measuring change in fluorescence properly for those wells. Also, see what happens if you just select your treated samples for a melt curve graph. Your control samples have really high expression and it might be compressing the & axis.
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