Calculating Tm - Help Needed

Nigel McMillan nmcmillan at gpo.pa.uq.oz.au
Wed Jul 19 23:18:30 EST 1995


HARDIES at THORIN.UTHSCSA.EDU wrote:
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>From: sywang at whale.st.usm.edu (Shiao Y. Wang)
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: Calculating Tm - Help Needed
>Date: 15 Jul 1995 05:13:26 GMT
>Organization: University of Southern Mississippi
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>
>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. If Na+ conc is 0.05 M and the oligo is 50% G+C, I get
>
>Tm = 81.5 + 16.6(log 0.05) + 0.41(0.5) - (600/20) or
>Tm = 81.5 + 16.6(-1.301) + 0.205 - 30 or
>Tm = 81.5 - 21.6 + 0.205 - 30 or
>Tm = 30.1
>
>I've been doing PCR using an annealing temp of 60C so the 30.1 appears to
>be in error. Thank you very much for any help.
>
>Shiao Wang
>University of Southern Mississippi
>sywang at whale.st.usm.edu
>


Most people calculate oligo Tm using the formula

Tm = 2(A+T)+4(G+C)

Some also substrate 5oC from the final answer.  As you can see the 
Sambrook formula doesnt work for small DNA fragments.  Besides in PCR the 
acutal result is all that counts no matter what the calculated Tm is.

Nigel McMillan
University of Queensland
AUSTRALIA




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