Hi everyone,

It may not be a problem really but...I was very surprised because although in manuals and from my experience, I know that the Ta can differ up to 5 degrees or more from the Tm of the primers, it is the first time that I found a Ta higher than the Tm of them. They have Tm 59.60 and 59.66 (%GC 41.67 and 55) each one. I checked it with online software and obtain 52ºC for them but when I run in gradient (from 49 to 65ºC) for optimizing, the best Ta was at 64ºC.

The shape of the amplification curve is normal, as the melting curve, and efficiency is 100.12% (R-squared 0.9984) and I have not other products of amplification.

What do you think? Thanks in advance!

Ángel

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