"high stringency" washes -NOT?
David L. Haviland, Ph.D.
dhavilan at IMM2.IMM.UTH.TMC.EDU
Wed May 7 09:40:34 EST 1997
At 12:09 5/4/97, Jim wrote:
>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).
Jim (Dr. Graham):
Me thinks I have an answer. Where did you get the formula? Knowing you
and desire to be precise, I don't think you typed it wrong. However, it
may have been printed wrong...
A Log10 of a number less than 1, produces a negative number. So from some
rusty rules of algebra, if 16.6 times a negative number is negative, its
subtraction would be positive and would add the number to the 81.5. With
the formula above, I would agree with your conclusion that the more salt
drops the Tm, so at a certain temp, more salt would mean less hybridization
and cross species hybridizations would be hard to do. This isn't how it
works in practice.
I think where you got the formula is wrong. I know of this one from Davis
et al. The little black spiral bound mehods book that has a few recipes
that are outright wrong (PBS & TBE). However, this formula appears correct
as I've seen it elsewhere and I've used it religously for RNAse protection
assays. I believe it is around pp 75-76.
I have it as:
Tm = 16.6Log[M] + 0.41(%G/C) + 81.5 - Pm - B/L - 0.65(%F)
Where:
M is molar concentration of Na+ (1XSSC is .165M Na+). %G/C is the percent
G and/or C bases in the probe. Pm is the % mismatched bases. %F is the
percent formamide. B is 675 for probes up to 100 bases and L is the length
of the probe in bases. Notice the 16.6Log will end up being a negative
number by itself for Na+ concentrations less than 1M. For long probes,
over 700 bases, in practice the formula reduces to:
Tm = 16.6Log[M] + 81.5
For large probes the %G/C becomes a ever smaller factor. For Southerns (at
least for me) formamide isn't used. The % mismatch isn't usually known in
cross species (or homologue) hybridizations. Finally the B/L is moot with
large probes over 675 as it approaches 1 as L increases.
Now, 1XSSC is (.165M): Tm = 16.6Log[.165] + 81.5
= 68.5 'C
With 5XSSC (.825M): Tm = 16.6Log[.825] + 81.5
= 80.1 'C
The way this one is written, the rule (and practice) hold.
Hope this helps,
David
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