Maximum Annealing-Temperature for Real-Time? - (Nov/22/2011 )
Hi, This may be a rather stupid question, but i'm just not totally sure:
For generating primers for my real-time qPCE assay I used a primer-design software that was recommended to me (forgot which one it was just now, but it isn't important here really). I specified melting-temperatures around 60°C for each primer, as this was recommended too. However the Program seems to have used an inaccurate algorithm for calculating the meling-temperatures, as the resulting primers have melting-temperatures around 80°C! (as shown by assays made by the manufactor).
So a good Idea would be to use rather high Annealing-temps in the qPCR reaction as well, yes?
However not only do most protocolls recommend annealing-temps at 60°C, but the manual for my qPCR-kit (Absolute Blue by Fischer-Scientific) also states Annealing-temps from 50-60°C can be used.
Since the Elongation-Temperature for this kit is at 72°C, i would think that i could anneal at upto 72°C as well. Is there any reason to restrict annealing to 60°C for real-time?
What are the sequences? There are many ways how to calculate Tm, some give very very different results. May be your primers will work on 60 just fine. Give it a try.
I stick with the calculations I've tried, like the one in primer3, these work fine with me and I don't care what the manufacturer writes.
well the reactions do work at 60°C but i fear that might be a little suboptimal, if not a little unspecific. The efficencies of the reactions are rather low (82%-90%), and i have a very small extra lump in the melting-curve which might be weak primer-dimer-formation.
I usually used to design my primers in Gap4, which always gave melting-temperatures close to the temperatures stated by the manufactor later. But sadly Gap4 does not state the formula it uses to calculate these temperatures.
Since every calculation method yields totally different melting-temperatures, i prefer to stick to the experimentally proven melting-temps. (thermo-fisher does melting-curves for every synthesized primer).
Of course the reactions should basically work at temperatures far below the melting temp (as the primers are already denatured by the 95°C step in each cycle, and the annealing should go easier the lower the annealing-temp), but i think its probably better to stick close to the actual melting temp, especially because i'm afraid the relativly low temp. may increase primer-dimer formation.
Well the Tm is nice in theory, but the main goal it to get qPCR working, even experimental Tms may not be determining for beaviour of the reaction. If you have extra peaks on melting curve, you should try increasing the Ta. At the same time look at the Cts, if they don't go too high.
What Tm does primer3 gives for your primers?