I am attempting to reproduce results from a number of journal articles all referring to the same SNP. In doing this I'm using the same primer set outlined in the articles. When I attempted a run the other day I experienced what I can only assume was primer dimer (~40bp product, bands not visible at expected bps.) While the primers and restriction enzyme were the same among numerous articles, the annealing temperatures in the reaction varied a fair amount (52-59C). All were above the individual Tms of the primers which struck me as odd.
Calculating the Tm via primer blast here are the numbers:
Forward Tm: 50.14 [5'-GCTCTACTTCCTGAAGACCT-3']
Reverse Tm: 51.67 [5'-AGTCTCACTCACCTTTGCAG-3']
Other sites gave slightly higher numbers (54-55) but they are still below the 57C that I used in my reaction. I do not have access to a gradient thermocycler otherwise I would have tried a range myself.
If an annealing temperature is chosen above the individual Tms how do the oligos have a chance to anneal with the template and eventually extend? I remember hearing the Tm calculation is more involved and includes finding where the melting curve changes direction. But on the surfance I would think the primers would never have a chance to anneal at such high temperature? What is wrong about my interpretation?
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