hello, I am a biochemistry student and I'm trying to optimize a qPCR assay.
I have newly designed primers and they should amplify a 150 bp piece of DNA ( checked by software). I have amplification in the qPCR but is there any way to verify if that amplification is indeed from a 150bp amplicon?
Thanks in advance
The simplest solution is to run a 2% Agarose gel with your qPCR product and DNA ladder. If the amplified curve is indeed 150bp then you will see the band.
Have a great day!
You should run the gel but you can't use the qpcr product if you run melt curve function.
Since the melting is related to the nucleotide composition, you could get an estimate given:
Tm= (wA+xT) * 2 + (yG+zC) * 4
we could say:
Tm = x + z
but then you have an equation with two incognita. However, you know the composition of the amplicon beforehand since you designed the primers. The Tm should be very close to what you calculate with the first eq. (or other more precise calculators).
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