"high stringency" washes -NOT?

Gene Huh gshuh at socrates.berkeley.edu
Thu May 8 10:24:53 EST 1997


In article <MacWeb04May97120914 at clarkcentris650.wustl.edu>,
graham at bionet.wustl.edu (Jim) wrote:

# Greetings colleagues,
# 
# Anyone else find it puzzling that "high stringency" washes feature a
reduction in salt, a component used during hybridization to lower the
annealing midpoint temperature (Tm)? One might reasonably expect high salt
in washes to help melt less stable mismatched duplexes by lowering the Tm
of all duplexes. Such a wash would be an undescribed "high salt -stringent
wash".
# 
# Apparently, "(mismatched hybrids) are more stable at high salt
concentration than low". This is contrary to the calculated effect of
increased salt concentration when using Bolton and McCarthy's: 
# 
# Tm = 81.5 - 16.6 (log[Na+]) + 0.41(G/C) -0.63(form%) -600/L
# 
# determination of melting temperature (which I described above).
.
.
.
# E-mail me copies of responses directly please,
# 
# Jim
# J. Graham PhD 
# Biology Department 
# Washington University of St. Louis 

According to the equations for Tm estimates of DNA-DNA, DNA/RNA and
RNA/RNA quoted by Sambrook and/or by the RedBook, the above equation has a
sign switched.  I think it should be "+16.6(log[M+]", since the
Bolton-McCarthy equation as quoted by Sambrook and several other equations
all have the cation component added rather than subtracted.  Needless to
say, the apparent paradox is no longer.

Hope this helps
Gene

-- 
Gene S. Huh, Ph.D.
Department of Molecular and Cell Biology
Life Sciences Addition, Room 221
University of California at Berkeley
Berkeley, California   94720-3200
Email:  gshuh at socrates.berkeley.edu



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