None are perfect. I would still do a gradient PCR around the predicted Tm to get the specificity.

You better check you primer carefully before set up PCR. But generally New England Biolab can give you good annealing temp prediction. -5C of Tm for annealing temp works fine. If you have gradient PCR it is easier, but if not just do a manual increase for Ann. temp. Like 2 C

good luck

Hi,

Try this by generating a free account and check the Tm, primer dimer and the secondary structure as well.

https://www.sigmaaldrich.com/technical-documents/articles/biology/oligo-evaluator.html

Cheers

You can use anyone of these to calculate Tm. But still u have to optimize the PCR conditions.

The most accurate ones are those using the nearest neighbor method. Try Primer Inspector:

http://www.molbiotools.com/primerinspector.php