Calculating Tm - Help Needed

Peter Myler mylerpj at
Wed Jul 19 11:50:16 EST 1995

On 18 Jul 1995, Shiao Y. Wang wrote:

> Roger Anderson (rogera at wrote:
> : This is the equation for hybridization of long probes >100 bp to even 
> : longer DNA. 
> Probably true but Sambrook et al read: "... predicts reasonably well the Tm
> for oligonucleotides as long as 60-70 nucleotides and as short as 14
> nucleotides."
> : 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. 
> I was actually using the K+ conc (50 mM) in the 16.6 log M. No Na+ added
> unless associated with the DNA. Mg++ is unaccounted for; although only 1.5
> mM, it may play a large role.
> Very true about the cookbook part. Makes science interesting and
> frustrating at times. 
> By the way, someone suggested I made a mistake where
> 0.41(fraction G+C) perhaps should be 0.41(% G+C) or 0.41(50) instead of
> 0.41(0.5). Sambrook et al. does say fraction and not %.
> There does not appear to be a simple solution to the Tm of
> oligos question. The best method seems to be the nearest neighbor method
> which is not easily done with a calculator. The software Oligo uses it but
> the last time I checked, it was $640. Anyone knows of a less expensive
> software that uses the nearest neighbor method? Primer is free but I'm not
> sure about the method used.
> Shiao Wang
> University of Southern Mississippi
> sywang at
GENERUNNER uses all three (thermodynamic nearest neighbor, %GC and 
2xAT+4xGC) methods for calculating Tm, so you can compare and pick the 
one you like.  They do not allow adjustment for [Mg+2], but do allow 
formamide.  The program does many other neat things as well and is fairly 
in expensive ($250).  It is well worth the time looking at a demo.  

I am not affiliiated with Hastings software, just a user.

Peter J. Myler                                 phone: (206) 284-8846x332
Seattle Biomedical Research Institute          FAX: (206) 284-0313
4 Nickerson Street                             e-mail: MYLERPJ at U.WASHINGTON.EDU
Seattle, WA  98109-1651

More information about the Methods mailing list