Melting Temperature of Oligos - (Jan/07/2008 )
Iam using nearest neighbor formula for calculating the Tm of shorter oligos:
Tm =dH /(dS+R x InC)
Where: dH is Enthalpy. dS is Entropy. R is Gas constant(1.987 cal/Kmol) and C is the Primer concentration. dH and dS is
calculated using Santa Lucia values. Salt correction is added while calculating dS.
ds (salt corrected) = [Na+] + 0.368 X N X In([Na+equivalent])
Na equivalent is calculated by using formula below:
Na equivalent = Na+ + 4 x sqrt(Mg++)
In some literature I came across the effect of dNTP on magnesium ion concentration. Till now I was not considering dNTP concentration for calculating the Tm of my oligos. The formula given in the literature taking in account dNTP concentration is:
Na+ equi= Na+ + 120 X sqt [(Mg++) ]- [(dNTP)]
Using this formula, I am getting Tm 10-15 degrees higher. Could any one suggest me the formula for calculating Tm using nearest neighbor formula and also taking in account dNTP concentration.
Could I please ask the question, why do you care? You will not, likely, be able to precisely get a correct answer, and very likely do not need one. It's not even clear there are good ways to measure it.
Good primer pairs work, bad ones do not, at any annealing temperature. Pay more attention to the hairpins and primer-dimer structures and far less to the Tm.
The units in the formulas you give above are not even consistent, so there are hidden constants which are measurement unit dependent in there somewhere.