RNase A preciptitation

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Jun 2 21:31:00 EST 2000


On Fri, 2 Jun 2000 18:24:52, "Neil McKenna" <nmckenna at ottawa.com> wrote:

ð We recently prepared come RNase A as a 100 ug/mL solution as described in
ð Sambrook, Fritsch and Maniatis  -- we made a solution at pH=5.2, boiled it,
ð then adjusted pH to 7.4. We had been using it for several weeks and found it
ð was degrading our DNA when used at high concentrations, so we decided to
ð leave it overnight at 65 C. Unfortunately, when we came in this morning, it
ð was all precipitated, and we cannot seem to resolubilize it.

WOW! YOu guys sure are hard on RNases -- must be an RNA lab! Seriously:
1- the initial stock is 10 mg/ml (not 0.1 mg/ml). Tris buffer at pH 7-8 should
work fine. You can't use phosphate buffer, as I recall -- that does
precipitate.
2- off the top of my head, heating for 10 min at 95C or 100C is long enough.
You must slow-cool afterwards to allow the RNase to renature (refold)
correctly.
3- precipitation after 65C overnight is likely to be denatured protein.
Unfolding and aggregation is a *kinetic* process. Also, if there were any
thiol reagent (DTT, BME) present, the RNase A will not refold correctly after
heat denaturation.
4- Best way to dissolve RNase A precipitate -- heck, best way to treat RNase
anytime -- is with SDS and proteinase K... (RNA labs, you know what I mean).

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