5 replies to this topic

[biojoe]

Hi everyone.  I'm sorry to make a thread about primer design as they pop up a lot, but I'm really confused about Tm and Ta.  I've read that Ta should be about 5 degrees lower than Ta, but I've also read that a Tm in the range of 52-58 degrees is good.  The qPCR program I run has an annealing temp of 63 and extension step at 72.  Why am I hearing different things about Tm?

Here are two contradictory design guides:
http://www.miraibio....mers-that-work/
http://www.premierbi...mer_Design.html

Thanks for the help!

[Trof]

There are several ways to calculate Tm. It's confusing, because it can be very different. But generaly you need lower annealing than Tm.
I design mine to have Ta 60, and use primer3 for optimal Tm around 64-65, so 5 degrees higher than Ta. But Tms from the primer specificaton from manufacturer comes says 10 degrees or so lower. You need to use one method and see what temperature difference best works for you.

[biojoe]

Trof, on 16 March 2012 - 08:21 AM, said:

There are several ways to calculate Tm. It's confusing, because it can be very different. But generaly you need lower annealing than Tm.
I design mine to have Ta 60, and use primer3 for optimal Tm around 64-65, so 5 degrees higher than Ta. But Tms from the primer specificaton from manufacturer comes says 10 degrees or so lower. You need to use one method and see what temperature difference best works for you.

Thanks for the response.  I'm still a bit confused though... why is there the discrepancy?

[Divya Pothula]

Try a Gradient PCR or best is a column PCR in order to understand the best annealing temperatures. I found out the best annealing temperatures for my primers through that method, as calculations were always a bit confusing.

[Trof]

biojoe, on 16 March 2012 - 08:24 AM, said:

Thanks for the response.  I'm still a bit confused though... why is there the discrepancy?

The primer-template Tm is a real experimentally observable event, that depends on many things, on concentration of the primers and template, amount of salts, dNTPs and so on. But you won't try all the primers in real experiment to estimate the Tm, so you need to calculate it. And every calculation is different, some only takes the  CG/AT content, like just an approximation, some also calculate with the salt amounts, and that's what differs in your reaction. There are at least two different methods for salt correction and for thermodynamics. Maybe some of them are calculationg with different salts or who knows, I don't even know amounts of these things in my mix, it's proprietary.

I have a mix with great efficiency over broad range of temperatures (yet not always, of course) and set my first try empirically four degrees less than Tm from primer3. In 90% of time the reaction just works, if not I try gradient, or aditives. In 1% of cases I can't do anything with it, as the primers are bad, and redesign.

[phage434]

I've noticed that people with little experience in PCR worry far too much about Tm and Ta.  If you design your primers in any sort of reasonable way, they will usually work with an annealing temperature of 55C.  If they don't, then try a gradient from 50-65.  If they still don't, then it is time to redesign the primers.