How does 1st BASE calculate the Tm of an oligo? Why does the Tm value from my own primer software differ from the Tm reading on the datasheet?
We use two standard approximation calculations. For sequences less than 14 nucleotides the formula is: Tm= (wA+xT) * 2 + (yG+zC) * 4 where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively. For sequences longer than 14 nucleotides, the equation used is (Wallace Rule): Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC) ASSUMPTIONS: Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0. There are up to 9 different algorithms used to calculate the Tm of oligos and the different Tm values does not indicate that the primers will different from one oligo service provider to another. Simply use the same algorithm or software to optimize the Tm of your oligos. Then perform your experiments as you designed it to be. In the process of synthesis, we don’t change anything with regards to the Tm of the oligos and the difference shown in the datasheet is just a matter of different ways of calculations.
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