Calculating Tm - Help Needed
Mark D. Garfinkel
mg16 at kimbark.uchicago.edu
Sun Jul 16 09:53:18 EST 1995
In article <3u7ipm$m63 at server.st.usm.edu>,
Shiao Y. Wang <sywang at whale.st.usm.edu> 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
>mistake?
>
>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
--
Mark D. Garfinkel (e-mail: mg16 at midway.uchicago.edu)
(c) 1995; all rights reserved. Permission granted for Usenet quotation
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