I am amplifying a gene where in a gradient pcr i am getting amplification at an annealing temperature about 5 degrees (67) higher than Tm (62.5)? What is wrong here? Also, I am getting a very strong intense band at wrong size for all the temperatures tried below Tm. Pls help
It is very common to get annealing at higher than predicted Tm. If it's a problem in your case, especially if you're getting mis-annealing at lower temperatures, consider using touchdown PCR to find an optimal temperature. Or, pragmatically, if you have any flexibility in primer design, simply order a set of primers and try them all. Primers are cheap today, and you'll often find a set that work even where the design programs claim they won't.
Tm of primers would not tell much about PCR conditions. When you get non-specific bands, it is worth to try higher temperature.
Your temp trying is already close to the temp to extend, so that shuttle PCR is available. Shuttle PCR has just 2 steps: 94 to 96 degrees for melting DNA and 68 to 74 degrees for annealing and extension together.
You might want to add DMSO or betain. They could help to reduce mis-annealing. If the non-specific bands are smaller and longer than bands you expect, longer and shorter extension times may give you better results, respectively.
And remember, primer design is also important. Check you have sequences repeating in your template and avoid to design primers from such sequences.
Regarding higher annealing temp than Tm, I often use T7 primer of which Tm is 48.3 degrees. The primer works well at 55 degrees of annealing temp. Usually, primers are excess in PCR reaction tubes, pushing the chemical equilibrium toward annealing. That is what I speculate.
The TM of the primer is also influnced of the salt concentration of your mix. Do you try the PCR gradient? Or you try the calculation of Tm and take in consideration the salt concentration. http://www.biophp.org/minitools/melting_temperature/demo.php