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
A lot of the difference comes from the situation where Tm and Ta are calculated at different salt ( Na and Mg concentrations) because historically Tm was more used in probe hybridisation in Southern Blotting Since both are salt concentration dependent there will be a lot of variation in both depending on the amount of salt. It is usually best to use a calculation specifically for PCR when the salts have been allowed for in the calculation of Ta and even better to run a temperature gradient of annealing temperature because that gives you not only a clean product temperature but also a good amplification temperature under the conditions that you are using.
The terms come from the temperature of melting of pre-existing duplex - Tm or temperature of hybridizing new one in annealing process - Ta. The last corresponds for primer annealing better because of the nature of the process.
Hi Angel
All calculations for Tm or Ta are pretty much predictive. Most of times very help, but the reality depends on your real reaction results. If you don't see any problems, then it is OK.
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