Calculating Tm - Help Needed

[HARDIES at THORIN.UTHSCSA.EDU]

On 15 Jul 1995, 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?
> 
> 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.
> 
And Mayumi Yagi replied:

: The version I have differs from yours in that the factor is 0.41(percent 
: G+C), not fraction G+C.  This would change your calculation to 81.5 -21.6 
: + 20.5 - 20 = 60.4.

Almost right; except the last term is, of course, -30 giving a
calculated Tm of 50.4.  The remaining stability comes from the Mg in
the PCR buffer. For 1.3 mM Mg you get 8-10 extra degrees of stability;
for 2.0 mM you get about 16 C; etc.  To my knowledge, neither the
equations in use, nor any of the programs in use account for the Mg. 
NBI's oligo [no affiliation] does an empirical correction to obtain a
"optimal annealing temp." that crudely takes care of it, but they
don't recognize that the amount of Mg matters and sort of have it
confused with the GC rich template problem. I should say that I
haven't seen their most recent version, so maybe they've improved it. 

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu