for example, we designed a new primer pair which is specific to coat protein of a particular plant virus. and its melting temperature is recorded as follows:
Forward: 62.2 C
Reverse: 60.6 C
So, in general we follow a rule that "average of those two temperatures (61.4 C) and do a gradient PCR from 58.5 to 64.5 C" and by this we used to predict the annealing temperature (ta).
is there any software tool to predict ta or something else. please help us to find it.
There are many in silico programmes that will give you annealing temperatures but the best way is still to run a temperature gradient of annealing temperatures on a gradient pcr machine which will show both the strongest amplification and also the cleanest amplification
Hello,
You absolutely right in following the conventional method of finding out the annealing temperature. However, if you want to use available tools to get the desired annealing temperature then you can use https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
You can try a Tm predictor, usually the company you purchase the primers from will have one. I use IDT (integrated DNA technology)
Really, just trying out the primers to see if they work is the best option. If you can do a gradient that's great, otherwise, your solution of just trying the average is a good one
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