RNase decontaminating soultion.

Rimas Rimantas.Plaipa at gf.vu.lt
Wed Feb 9 08:02:14 EST 2000


"Kajetan H. Groicher" wrote:
> 
> Hey all-
> 
> I'm wondering if anybody has a 'in-house' recipe for an RNase inactivating
> solution.  I have in the past used RNase away or similar products for
> decontaminating tip boxes and gel units etc of RNases.  A common protocol
> source recommends treating gel boxes with NaOH before use to remove
> contaminating RNases.  The bottle of RNase away indicates that it is an
> alkaline solution, and it seems to foam a bit.  So I got to thinking....am I
> really paying money for a NaOH-SDS solution that I can make my self for
> cheap, or is there more to it?
> 
> Not a real urgent or pressing issue, but thought someone may have some
> insight.
> 
> Kajetan


Once I'd performed experiments to test the efficiency of various
procedures to kill RNases. The results were posted to this newsgroup
also (autumn '96, I think).

NaOH+SDS indeed may be used as inactivating solution, but its 'power' is
not instantaneous - the half life of pancreatic RNase (which was used in
these experiments as a model) is about 6 min in 100 mM NaOH + 0.1% SDS
at 18 C. Without SDS inactivation proceeds much slower (half-time about
30 min) but at higher temperatures (30 C) SDS is no longer required.
Presumably it helps to denature the protein and thus makes cystin -S-S-
bridges more susceptible for attack by hydroxide because at low
temperature alkaline conditions alone are not enough to unfold the
globule. There are some reagents which also attack -S-S- bridges and
effectively inactivates RNases in alkaline media - various
reducing/sulfhydryl agents (ME, DTT, sulfite, thiosulfate,
thiophosphate, thiourea etc.) but their presence can make more trouble
if the solution will not be washed completely away. NaOH alone is quite
harmless if the things are to be used with solutions of reasonable
pH-buffering capacity.

At that time I'd attended certain meeting where 'RNases away' was handed
for free but I didn't take any - and much regretted later about this. I
certainly would have made tests about its real efficiency and
composition / mechanisms of action.

Some of the aforementioned agents (thio-compounds) may be easily
detected by spotting the small drop on the undeveloped photo/X-ray film.
If they are present the spot will immediately turn black or brown. Maybe
someone who have that solution can perform such an experiment and mail
the results to me - or to the newsgroup if one is not afraid to find
oneself  in the black lists of the biomedical industry. (UV spectrum
would be very handy also).

I also have some reserve about the overall usability of such a solution.
I use NaOH+SDS to wash big objects - tweezers, spatulas, buffer tanks of
PAGE apparatus (with the addition of some sodium pyrophosphate which
helps to prevent irreversible sticking of 32P). I never wash Eppendorf
tubes before work - they have to be clean (and usually are) due to the
automatic process of production. If you clean something with something
harsh you have to wash away the cleaning agents and the dirt you wanted
to wash away may be reintroduced again. The best way to prevent
contamination is not to touch the thing at all.

-- 
Rimantas Plaipa,

Department of Biochemistry and Biophysics,
Vilnius University,
Ciurlionio 21/27, Vilnius 2009, Lithuania.

E-mail: rp010gf at voruta.vu.lt
Phone: (370-2)-650381 
Fax: (370-2)-236660
---




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