Protocol Online logo
Top : Forum Archives: : Molecular Biology

Contradicting Tm - (Jan/28/2007 )

I pasted the nucleotide sequence of my primers into the oligonucleotide calculator in the Biotool when I was designing my PCR primers. The Tm resulted from the program was pretty satisfactory, in the range of 56-60 oC.
However, when I received my primers from the company, the Tm for my primers were basically far lower than what I got from the oligonucleotide calculator in Biotool. They turned up to be in the range of 50-53 oC.
Why????????? I am very curious to know the reasons behind.
Thank you.


well there are several method of calculating tm

base stacking
salt adjusted

I am sure there are several ways to calculate each method.


The only "true" way to know a Tm is by experimental testing. The programs are just predictions based on various algoritims, but generally give a good idea of the approximate Tm. I have seen these prediction programs vary up to 10 degrees in the calculation of a primer Tm.


Thank you for your replies, perneseblue and tap14.
Now I understood.
And I also managed to get my PCR product from doing gradient PCR this morning. Apparently, the Tm gave by the company is correct. smile.gif