Dear vikashini,

1) Annealing temp. is 50c less than the Tm. eg. if Tm is 63 then annealing should be 58 0  c.

also use this formula to calculate.

2) annealing temp = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 25.

3) Also you can use software to calculate Ta, primer 5 is better.

Thank u Mr. Suhas. In my case the Tm is 52 but am getting a good amplicon only at 58 degrees. Y is it so? Will it be reliable at this temperature.

if you are getting PCR band at expected size then confirm you PCR product by sequencing

We will send the pcr product for sequencing but before that I should ensure that the product is pure with no primer dimers.