primer melting point?

nvahsen at hot.hgmp.mrc.ac.uk nvahsen at hot.hgmp.mrc.ac.uk
Tue Nov 23 14:59:05 EST 1999


On Tue, 23 Nov 1999 19:02:18 GMT, daniboyton at my-deja.com wrote:

>Can anyone please tell me what the melting points for GC and AT pairs in
>primers are. I believe they are 2 and 4 degrees respectively but I could
>be wrong.                            Thanks
>
>
>Sent via Deja.com http://www.deja.com/
>Before you buy.

You mean the Wallace/Ikatura rule for short oligos (~<25 bp)
Tm = 2°C(A+T) + 4°C (G+C)

cited in: Wetmur, Crit Rev Biochem Mol Biol 1991, 26:227

It was (afaik) originally applied to 1M NaCl hybridization reactions.
However, it works also for Tm estimation for PCR primers. PCR is very
robust against primer Tm. For better estimates of Tm you must use the
nearest neighbor (n-n) model based calculation that also considers
oligo-concentration and sodium equivalents.
A colleague of mine and I have programmed a excel-spreadsheet
application for these n-n calculations. Maybe this can help you. You
can read about it in the upcoming december issue of Biotechniques (E.
Schutz & N. von Ahsen).

Best regards

Nicolas





More information about the Methods mailing list