Calculating Tm - Help Needed

Shiao Y. Wang sywang at whale.st.usm.edu
Tue Jul 18 16:51:39 EST 1995


Roger Anderson (rogera at atcg.com) 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 whale.st.usm.edu





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