Calculating Tm - Help Needed

Shiao Y. Wang sywang at whale.st.usm.edu
Sat Jul 15 00:13:26 EST 1995


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




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