Melting

... However, I would disagree about the microwave, we use it standardly for melting agarose and agar should work as well. One just has to check that it doesn't boil. It's always good to mix the bottle during heating. By that ...

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Not sure what you want, but look at this: http://www.biophp.org/minitools/melting_temperature/demo.php?formula=basic

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... protein, other than the propensity for helices being sequence dependent. Relate it to the coil-helix transition, fractional helicity and melting temperature.

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Your primer melting temps for each set are just too far apart. Just consider that at say annealing temp of 59, only 50% of one primer is bound to template and ALL (or close to 100%) of the second primer is bound non-specifically ...

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... as populations of molecules. Each primer is a population of molecules, and each strand of the template DNA is a population of molecules (after melting of the template DNA). Any one molecule of primer may stick anywhere on a molecule of template DNA whenever hydrogen bonds can form. The duration ...

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