Dear Ranjith,

You can calculate annealing temperature using this online tool.

https://eu.idtdna.com/calc/analyzer

SD

Hello Ran

I have written and advised much on this subject; Indeed find attached a word document describing the basics of good primer design, including the question you pose.

To answer your question directly, having calculated the melting temp of your primers using the above tool (which I have also cited in the document provided) try an annealing temp of Tm-2C and make sure that your forward and reverse primers do not differ in terms of their Tm by > +/-2C 

In terms of a permissive temp range within which you can design and successfully use such primers any thing from 55C to 70C is optimal and then as mentioned try 2C below that temp for your actual PCR. Thus, I tend to design primers with a Tm approximating to 60C most of the time although for GC rich targets you might need to go up to 70C; hence the large permissive range

For primers with a Tm of 55C for example necessitating a Ta of approx. 53C perform a 3 step PCR, i.e.

Denaturation: 95-96C for 5 to 20 sec (depending on polymerase and GC content; try a higher temp and longer time for target regions with GC content > 60%)

Annealing temp: TM-2C, i.e. 53C for a Tm primer of 55C

Extension: 72C for 10 sec (100-300bp) to 1 min (up to 1kb)

For primers with a Tm of say 65-70C you can combine the annealing and extension cycles into one, i.e.

96-98C for 5-20sec

65C for (up to 1 min) for Tm primers approximating to 65C-70C

Both work very well all things being equal

In addition, I would recommend designing 2 sets of primers for your target

Finally, when you calculate the Tm using a tool such as IDT (which is one of my preferred tools) remember to include things like your Mg and dNTP concentration as well as your primer concentration all of which can affect your Tm.

Hope this helps

Thank you Dr. Sibnarayan Datta and Mr. Laurence Stuart Hall for your valuable suggestions. I will look into it.

Thank you once again..