formamide/ membranes for hybridizations.
wrp at cyclops.micr.Virginia.EDU
Tue Nov 5 11:44:59 EST 1991
Regarding formamide for hybridization and blotting membranes.
As the poster surmised, formamide reduces the Tm of DNA-DNA,
DNA-RNA, and RNA-RNA hybrids. For DNA-DNA, the effect is 0.7oC/%
formamide. So why not do hybridizations at room temp in 60%
formamide? Because, in addition to reducing the Tm, formamide reduces
the nucleation-rate-constant for reassociation, so that the reaction
would take 5 - 10X as long to hybridize to an equal extent. How do
people get away with using formamide? Either they (1) have such a
high concentration of probe (or DNA on the filter in the case o
plasmids) that it does not matter, or (2) they use a accelerating
agent, such as dextran sulfate, which provides a 10X speed up, or (3)
their blots don't work very well. For genomic blots, dextran sulfate
will let you use formamide and still get good results. The problem
is, especially for nitrocellulose, dextran sulfate + formamide can
cause horrendous background problems.
We routinely do genomic southern blots and northern (RNA)
blots at 65oC with no formamide, no dextran sulfate, hybridizing for
40+ hours with excellent results. I do not believe there is any good
reason to use formamide, especially now that nylon membranes are
available that are not affected by high temperature. Most people who
use formamide do not realize that it slows down the hybridization
reaction, so that usually there is a net decrease in hybridization.
Re: nylon vs nitrocellulose. Nitrocellulose has a marginally
higher capacity for DNA or RNA, this can be important for dot-blots and
slot-blots. Otherwise, it is much more fragile (it tears very
easily), it ages very poorly, it is more difficult to handle (wetting
can be difficult), and it is much less reproducible from lot to lot.
I blotted for 5+ years on nitrocellulose before changing to nylon (AMF
Cuno's Zetabind or S+S Nytran) and I would never go back. No baking,
no tearing, no background, its great.
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