Read the DNA sequence from one end to the other
& add up the DNA strength parameter (Given in table 1).
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Calculate the strength parameter per base (E) as: E = DNA strength parameter ⁄ Length of sequence
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Substitute the value of E, the length of sequence (which is 70 basepair in this case), the Na^{+} ion concentration and the nucleotide strand concentration in the given equation. Tm (°C) = (7.35 × E) + [ 17.34 × ln(Len) ] + [4.96 × ln(Conc)] + [0.89 × ln(DNA)]
 25.42
This gives the melting temperature for a 70 bp window.
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The above procedure is repeated for the complete sequence with a stepsize of one bp.
Figure:Flow chart to calculate the melting temperature
