Dear Gun

• gradients typically go from Tm-5C to about the Tm in 1c intervals
• Thus in your case I would opt for 59C to 64C

Dear Guna Ji

You can use gradient PCR from 56-64 C, generally we're selecting annealing time on the basis of Tm ; if your primer Tm is 63.5, so Tm-5 C, you can select annealing temperature.

As your FP having Tm= 63.5 and RP Tm= 64.5 , therefore, ideally annealing at 55 is the best option. You can set your gradient cycle either 2 or 5 degree interval and consider your annealing temperature range in between 50-63 degree.

I have had primer sets working well above their Tm so I would run a gradient of 59-69 expecting only 69 and possibly67 not to work. Primers are in huge excess so expect some primer to anneal and work even above the Tm