re-probing nylon filters
schalkwy at callisto.lif.icnet.uk
Sun Nov 27 08:49:55 EST 1994
I wonder if anybody has any ideas about the best way to strip
blots and store them between uses.(In my case it's Hybond N+
spotted with PCR products) For many projects, including the one
I'm doing, it makes quite a difference if filters can be
hybridised 30 times rather than 15. Filters can last a long
time, especially if they have a lot of DNA on them, but they
do occasionally die. It's not easy to pin down the
important variables, because problems happen sporadically,
and "torture test" experiments have to be very long to
simulate the whole useful life of a filter.
Common stripping methods include boiling in low salt solutions,
lower temperatures in 50% formamide, or alkaline treatments,
all with or without SDS.
Fashions in storage have run the gamut from
4 degrees in a vacuum dessicator (from the days of nitrocellulose)
through air dried or
damp in saran wrap at room temp (probably the most common)
to storage in prehybridisation solution.
What is your experience? Favourite methods? Particularly
interesting are cases where you have had blots die and think
you know why. My current preference is to strip using 50%
formamide, 40 mM sodium phosphate pH7, 0.1% SDS at 65, rinse in
the same buffer without formamide and store dry.
Some discussion-starting thoughts:
SSC-based recipes: citrate is neither a very good buffer at pH 7
nor much good as a chelator. 0.1 x ssc is pretty much unbuffered
and the solution will be pH about 5. This might be tough on DNA,
especially at stringent wash/stripping temperatures.
SDS: SDS is a nuclease inhibitor, but as a preservative for storage
it may not prevent bacterial growth. Many bacteria, E. coli to give
a banal example, tolerate high levels of SDS, and could be a source of
unfriendly enzymes (see below).
Nonradioactive detection methods: what is the impact of these methods
on filter reusability? Not only with respect to getting dyes
and enzyme conjugates off the filter, but what about the incubations
in low salt, mg++ containing (ie nuclease-friendly) solutions?
Imperial Cancer Research Fund
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