Calculating Tm - Help Needed

[Roger Anderson]

sywang at whale.st.usm.edu (Shiao Y. Wang) 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?

This is the equation for hybridization of long probes >100 bp to even 
longer DNA. THe equation for oligos in normal htb. conditions is Tm=93 - 
(600/L). However PCR conditions are quite different from hybridization
to a filter. We use the above formula and then test at +5 and -5 degree 
incraments. I believe there is a PCR formula but I don't have it handy.

>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 0.05M 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
>
You also did not count the K+ salt. The 16.6 log M is for monvalent 
cations and there are a few in PCR buffers. This is why molecular biology
is not like cookbook science. 


Good Luck,
Roger Anderson
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