Calculating Tm - Help Needed

Mark D. Garfinkel mg16 at
Sun Jul 16 09:53:18 EST 1995

In article <3u7ipm$m63 at>,
Shiao Y. Wang <sywang at> wrote:
>I'm using the equation in Sambrook et al. 1989 (pg 11.46) to calculate the
>Tm of oligonucleotides. My results don't look right. Can someone spot my
>The equation is: Tm = 81.5 + 16.6(log[Na+]) + 0.41(fraction G+C) - (600/N)
>Where N = chain length.
>My oligos are 20mers.

	Misapplication of the formula. The formula *you* used is for
solution hybridization with real DNA (i.e., probes of several hundred bp).
Usually this formula also includes a term for the effect of formamide,
which you have neglected to quote.

	For synthetic oligomers try:

	Tm = (2-deg-C for each AT bp) + (4-deg-C for each GC bp)

	Your 20-mer that's half AT would then have Tm = 2*10 + 4*10 = 60-deg-C,
in accordance with your own use. Note that this formula doesn't account for
nearest-neighbor effects, or for 3'-end "breathing" problems. Sometimes you
just have to do the experiment & systematically vary relevant parameters to
optimize PCR conditions for each primer pair.

Mark D. Garfinkel (e-mail: mg16 at
(c) 1995; all rights reserved. Permission granted for Usenet quotation
with attribution.

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