Calculating Tm - Help Needed
Wolfgang Schechinger
u7k0201 at sunmail.lrz-muenchen.de
Mon Jul 17 12:59:00 EST 1995
mg16 at kimbark.uchicago.edu (Mark D. Garfinkel) wrote:
>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
>with attribution.
>
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