EtBr in gels

Andre Hamel hamel at ccu.umanitoba.ca
Sat Jun 19 03:48:08 EST 1993


In article <9306182109.AA13410 at net.bio.net> NWALKER at JHUHYG.BITNET (Nigel Walker) writes:
>EtBr in gels not leaking out?!!.Whenever you run a gel containing the EtBr it
>migrates in the opposite direction to the DNA and hence into the buffer chamber
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^       
>
>Mind you..Ive always incorporated EtBr into the gel directly except for gels
>that run a long time....which tend to need abit of extra staining to view small
>quqntites of DNA.

Hence we always include at least 50 ng/mL EtBr in BOTH agarose gel AND in
anodic buffer (the end of tank that DNA will migrate towards) ... thus we
add 10 uL of 5 mg/mL EtBr stock per 100 mL of gel, likewise for cathodic
end of buffer tank, after gel has been submerged in tank, we add 10 uL
of same EtBr stock per 100 mL of buffer. ... 
This minimizes/eliminates the phenomenon of top of gel fluorescing more than
bottom due to EtBr having migrated out of bottom towards top (top being
where sample loading wells are, ie cathodic end) ... all we usually need
to do is to destain for few minutes in excess dH2O with mild shaking, in
order to reduce salt conc (which inhibits EtBr fluorescence/binding) and
excess EtBr, which if you didn't rinse, you'd see "leftover" on UV light
box ... 

likewise, for disposal, we prefer NOT to discard ANY EtBr containing
solutions/gels down the drain or into trash cans, dilute or otherwise (why
not at least try to give the environment a chance to recouperate) ...
simply follow the procedure in the 2nd edition of Maniatis, page E.8,
volume 3 ... use of hypophosphoric acid and sodium nitrate (final pH is <
3.0) ... container containing EtBr liquid waste (10x vol. tap H2O is added
to gels, autoclaved, then added to liq. EtBr waste prior to acid treatment)
... in hood ... after at least couple days (over weekend), if UV
fluorescence is not detectable (check a large clear glass flask full of
treated waste, unless waste is already in such), then excess sodium bicarb
is added, finally waste is simply poured down drain with excess water
flushing for couple hours after last amount of waste has entered drain ...
our chemical safety officer assures us this is better than him handling
both liq and solid EtBr wastes, only to be buried in landfill site to be
detonated for burning ... we could only imagine what potential
contaminating substances/"nasties" can end up in water tables/soils ...
(& food chain!?!) as result of such practices ... so if one can take
tiny "extra" bit of time to use one's well trained imaginations ....
I'll leave rest to ones imagination :-)

(ugh, I feel need to ... too late, here it is ...
If "leftovers" from unrinsed EtBr stained gel this is not washed off light
box, will dry and resulting powder (enough being produced via many users
over months, years of use, ought to amount to unacceptable accumulated EtBr
contamination .. perhaps in the milligram range ... simply use hand held UV
light, in darkened room, to visualize areas where EtBr has been used ...
I've encountered MANY a surprise, am sure there's MANY out there in
"netter-netter land" with similar experiences ... furthermore, if safety is
not enough incentive, economics wise ("hit 'em in pocket book ...", eh?),
excess salts leftover on light box filter from UNRINSED gels, once dried,
will surely lead to damage of there VERY expensive filters (worth almost as
much as retail cost of new UV transilluminator) ...

so to all "new" mol bio researchers (grad, summer students, etc) ...
please (listen to what you're mother says) ...  take note of your technician's
safety recommendations, a clean lab is safer and more productive place to
work (less chance of errors due to sloppiness/contamination ... let alone
"peace of mind")  8>} ... is this why most "head" techs (ones that
actually "run" the lab) are so stressed out/greying/ageing?

Good thing, eh?, that P32 and EtBr can be fairly readilly monitored/detected
... if in a "sloppy" lab, I'd be more anxious about "nasties" that aren't as
detectable (can't be smelt, seen, tasted or heard, but whose effects
might surely be felt later on ... :-)

Regular lab/suggestion/"gripe" meetings/sessions (in pub over few brew
tends to lead to less inhibited/more open & frank repart ... beats leaving
notes/signs as reminders left by those more conscienscious (obsessed?) of
responsibilities and courtesy towards (others) fellow lab "mates".

Does all this sound "too" familiar ... happens in "every" lab, except
those inhabited by non humans (perhaps from distant, more advanced cultures).

:-)

hi yo,
Andre
                            ********************
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
Infectious & Genetic Disease                     FAX: (204) 945-8062     
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
                            ********************



More information about the Methods mailing list