ETBr in Loading Buffer
D Picton
is not added to Loading buffer? I've never run across an article where
Ethidium Bromide is present in the Loading Buffer, it's allways
present either in the gel or as a post electorphoresis step.
D Picton
Krzysztof W. Wasowicz
wrote:
I tried using EtBr in loading sample when running PCR reactions. The
staining was poor and EtBr seemed to affect migration of my samples.
However, I use EtBr in the sample when I run RNA preparations in
standard TBE agarose gel. Works like charm.
Krzysztof W. Wasowicz
Department of Animal Anatomy
University of Warmia & Mazury
ul. Oczapowskiego 13 (Bldg. 105J)
10-957 Olsztyn-Kortowo
Poland
Phone: +48-89-5233688
Fax: +48-89-5234986
e-mail: waso...@moskit.uwm.edu.pl
ICQ: 58434323
Michael Witty
> On Mon, 04 Feb 2002 19:40:19 -0800, D Picton <pic...@bcc.orst.edu>
> wrote:
>
> >I was wondering if anyone has ever tried or knows why Ethidium Bromide
> >is not added to Loading buffer? I've never run across an article where
> >Ethidium Bromide is present in the Loading Buffer, it's allways
> >present either in the gel or as a post electorphoresis step.
> >
> >D Picton
> I tried using EtBr in loading sample when running PCR reactions. The
> staining was poor and EtBr seemed to affect migration of my samples.
> However, I use EtBr in the sample when I run RNA preparations in
> standard TBE agarose gel. Works like charm.
>
. . . I think ErBr intercalates, makes the helix untwist a little and this
affects shape and therfore migration. Also, if EtBr is just in the
loading buffer it would be depleted during a run and become separated from
the DNA - all leading to poor electrophoresis. Mike.
David L Haviland
> is not added to Loading buffer? I've never run across an article where
> Ethidium Bromide is present in the Loading Buffer, it's allways
> present either in the gel or as a post electorphoresis step.
Hi...
ALL THE TIME... I put EtBr in the gel (2-4ul/100mls) and I have it in
my loading buffer at 1ug/ml. I never use it in the running buffer as
we can't pour the buffer down the drain otherwise. It has to be
filtered through a EtBr reclaimation filter. I've never had a problem
in doing this over the last 10 years, neither in the running of nor in
the interpretation of the data. In fact, it was even a suggestion out
of BioTechniques, a millenia ago... I can't even remember the issue.
I even do it for northerns too but drop the amount in the gel to 1-
2ul/100mls of gel, lest the formaldehyde turn the gel into a "pink
flare" on the transilluminator.
Clear skies...
David
============
David L. Haviland, Ph.D., Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd., Houston, TX 77030
713.500.2413-Voice//713.500.2424-FAX
-----------------
If everything seems to be going so well, you have obviously
overlooked something.
============
---
Moshi Kotierk
I was under the impression that EtBr is positively charged, whereas DNA is
negatively charged. As such, if you load it onto the gel in the loading
buffer, the EtBr and DNA should go separate ways. If you look at a gel
that contains EtBr on a transilluminator after you have run it for a
while, you should see (at least, I usually do) a band at the bottom that is
less fluorescent.
Moshi
----------------------------------------------------------------------
Moshi Kotierk
Carleton University
Yesterday is history,
Tomorrow is a mystery,
Today is a gift,
That's why they call it the present!
----------------------------------------------------------------------
Sergio
David L Haviland wrote:
>>I was wondering if anyone has ever tried or knows why Ethidium Bromide
>>is not added to Loading buffer? I've never run across an article where
>>Ethidium Bromide is present in the Loading Buffer, it's allways
>>present either in the gel or as a post electorphoresis step.
>>
>
> Hi...
>
> ALL THE TIME... I put EtBr in the gel (2-4ul/100mls) and I have it in
> my loading buffer at 1ug/ml. I never use it in the running buffer as
> we can't pour the buffer down the drain otherwise.
Watch out, even if you add the EtBr in the gel only, it moves out from gel to
the running buffer due to it migrates to the anode. You shoul check your running
buffer before pouring it down.
Sergio
Michael Witty
>
> >>I was wondering if anyone has ever tried or knows why Ethidium Bromide
> >>is not added to Loading buffer? I've never run across an article where
> >>Ethidium Bromide is present in the Loading Buffer, it's allways
> >>present either in the gel or as a post electorphoresis step.
> >
> > my loading buffer at 1ug/ml. I never use it in the running buffer as
> > we can't pour the buffer down the drain otherwise.
>
> Watch out, even if you add the EtBr in the gel only, it moves out from gel to
> the running buffer due to it migrates to the anode. You shoul check your
> running buffer before pouring it down.
>
> Sergio
(sotto voce muttering) i still say etbr never hurt anyone. mike.
Michael L. Sullivan
Where I work, the level of EtBr used in a gel or in running buffer (on the
order of 10 ug per 100 ml) can be disposed of in the regular trash or down
the drain. Perhaps this is because of what the other Mike said, that is,
EtBr is not particularly hazardous.
Mike
>> Watch out, even if you add the EtBr in the gel only, it moves out from
>>gel to
>> the running buffer due to it migrates to the anode. You shoul check your
>> running buffer before pouring it down.
>>
>> Sergio
>
> (sotto voce muttering) i still say etbr never hurt anyone. mike.
---
Henk Veldman
Sergio schreef:
You can safely pour your running buffer down the drain after removing the EtBr by
absorbing it on activated charcoal or, more conveniently, with adsorption bags
(Amresco) that can accumulate up to 5 mg EtBr, enough for many liters of buffer.
Henk
--
H. Veldman
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)
Michael Witty
does anyone know what activated charcoal, and how to activate ordinary
charcoal? I have wondered about this for a long time. Mike.
Wolfgang Schechinger
> In article
> <Pine.SGI.4.33.0202061...@mole.bio.cam.ac.uk>,
> Michael Witty <mw...@mole.bio.cam.ac.uk> wrote:
>
> > (sotto voce muttering) i still say etbr never hurt anyone. mike.
>
>
> Compared with what else gets tipped down the drains . . . I'm inclined
> to agree.
Did people ever realize how much they shorten their lives by continuously panicking about the possible hazard of lab chemicals?
Happy Chinese New Year - welcome to the year of the horse!
Regards, Wo
Wolfgang Schechinger
IBMS
Taipei
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couic
"Michael Witty" <mw...@mole.bio.cam.ac.uk> wrote
> . . . I think ErBr intercalates, makes the helix untwist a little and this
> affects shape and therfore migration.
definitely yes; not recommended for the electrophoresis study of topoisomers
:)
Gerd Nilsen
Just remember to put some in the ladder too.
Benefit: beutiful pictures
Drawback: might be diff. getting equal amount in each sample ->
comparing amounts of DNA more inaccurate
And, yes EtBr interferes with DNA movement in gel, but there should be
no difference between adding the EtBr in the gel or in the loading.
Staining the gel after the run will always be the most accurate, but
also the most timeconsuming proc.
*Small fragments tend to loose to much of the EtBr during the run
(rem. always equlibrium betweeen bound and unbound EtBr, EtBr and DNA
runs opposite ways in the gel -> small bands run faster and loose
more)
On Mon, 04 Feb 2002 19:40:19 -0800, D Picton <pic...@bcc.orst.edu>
wrote:
>I was wondering if anyone has ever tried or knows why Ethidium Bromide