Boiling ethidium bromide gel
Jennifer Skerratt
Does anyone have any information (preferably not just gut feelings) on
how safe it is to boil the gel up in a microwave with the ethidium
bromide already in it. Does the ethidium bromide vaporise with the
steam?
thanks Jenny
Hiranya Roychowdhury
I do it all the time and EtBr stays put. This way, I've been able to reuse
agarose at least 5 times without having to add significant amts of EtBr. The
savings are substantial too. This, of course, is only for analytical purposes.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroy...@nmsu.edu
Vladimir Svetlov
In article <353fd...@newsroom.utas.edu.au>,
jenny.s...@postoffice.utas.edu.au wrote:
> Does anyone have any information (preferably not just gut feelings) on
> how safe it is to boil the gel up in a microwave with the ethidium
> bromide already in it. Does the ethidium bromide vaporise with the
> steam?
A good practice is to use a separate microwave for this and reheating food.
I'm not sure that anybody actually studied it but it's a good idea to keep
food away from the hazardous chemicals anyway. Recently I've taken up
adding EtBr to the loading buffer instead of the gel - far less EtBr to
worry about (in the running buffer etc.), lower background and you can boil
the agarose as much as you want.
Regards,
V.
--
Vladimir Svetlov, Ph. D.
McArdle Lab for Cancer Research
Dept. Oncology
UW-Madison
1400 University Ave.
Madison, WI 53706
un6...@genius.embnet.dkfz-heidelberg.de
On Fri, 24 Apr 1998 09:56:36 +1100, Jennifer Skerratt said:
>
>Does anyone have any information (preferably not just gut feelings) on
>how safe it is to boil the gel up in a microwave with the ethidium
>bromide already in it. Does the ethidium bromide vaporise with the
>steam?
EtBR probabyl won't vaporate by itself, but it will be carried along
by the water vapor. Almost any substance will.
Example: NaCl. Next time you have a cold, inhale some hot salt water.
helps you membranes.
clemens
Stephan
In article <svetlov-ya0240800...@news.doit.wisc.edu>,
sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
> Recently I've taken up
> adding EtBr to the loading buffer instead of the gel - far less EtBr to
> worry about (in the running buffer etc.), lower background and you can boil
> the agarose as much as you want.
Mmmm... I'm a little bit sceptical about that method. Can you really see
small fragments? (let's say, smaller than 1 kb?). What is the minimal
amount of DNA that you can detect? (let's say, can you see well all the
bands when you load a 0.1 ug sample of lambda/HindIII?).
If this method really works, I would be very interested to know more about
it (how much EtBr to your sample?).
Wolfgang Schechinger
Hi Hiranya!
You just boil your gel after taking the photo? - great! What
about an increasing background by DNA? is there any?
Wolfgang
> I do it all the time and EtBr stays put. This way, I've been able to
> reuse agarose at least 5 times without having to add significant
> amts of EtBr. The savings are substantial too. This, of course, is
> only for analytical purposes.
>
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Wolfgang Schechinger
University of Tuebingen
email: wgsc...@med.uni-tuebingen.de
http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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Dr. Duncan Clark
In article <6ht3df$m...@mserv1.dl.ac.uk>, Wolfgang Schechinger
<wgsc...@med.uni-tuebingen.de> writes
>You just boil your gel after taking the photo? - great! What
>about an increasing background by DNA? is there any?
I 've been doing this for may be the last ten years using TBE gels. What
happens over time is that the agarose gradually becomes more opaque and
less flexible (not due to just evaporation of water), more brittle. You
can reuse them at least 5x. Nowadays agarose isn't as expensive as it
used to be. background fluorescense from DNA is not a problem, possibly
because it is so dilute and sendly, boiling agarose will denature your
DNA. Giving that there is so little it probably never renatures so the
ETBr stains the ssDNA poorly.
Duncan
--
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk
Vladimir Svetlov
In article <REPLACE-2504...@macbb2212b.unil.ch>,
REP...@lbm.unil.ch (Stephan) wrote:
BIO101 actually sells such loading dye under the name of the Green Dye -
your normal ficoll-based stuff with EtBr in it. Inserts down to 200 bp
appear quite bright and on the plus side you don't have to run EtBr out of
the gel to reduce the fluorescent background, obscuring the small, low
intensity bands. When we ran out of the "green dye" I've started making me
own by adding 1/10 of the volume of EtBr to the sucrose-based BrPh/XyCya
dye premix. The bottle of EtBr I get it from dates back to the Reagan era
so I don't really know how close the 100 ug/ml is to the reality, but it
works OK as it is. I then added some EtBr (a pinch, as them French would
put it) into my 1 kb DNA ladder and I'm using it for couple of months
already without noticeable changes in bands appearance.
There are other methods to deal with the excess of agarose, such as cutting
up the gel into slices and keeping them at 4-8 C in a plastic bag with some
buffer. 1.5-2% gels can be stored like that for several weeks without
loosing the resolution capacity or EtBr. Gels with lower than 1% of agarose
can sometimes collapse the wells but normally they come right back at you
when you flood them with the running buffer. Some people store molten
agarose at 55 C but it's good only for a few days because a) stuff like
NuSieve does not take it very well and gel comes out very brittle and b)
them dumbasses forget about it and agarose with time turns yellow, brown
and eventually starts resembling coal. Then you have to boil it with NaOH
to get this shit off the glass.
un6...@genius.embnet.dkfz-heidelberg.de
On Fri, 24 Apr 1998 12:18:53 -0600, Vladimir Svetlov said:
>> Does anyone have any information (preferably not just gut feelings) on
>> how safe it is to boil the gel up in a microwave with the ethidium
>> bromide already in it. Does the ethidium bromide vaporise with the
>> steam?
>A good practice is to use a separate microwave for this and reheating food.
generally, I wouldn't use ANY laboratory instrument for 'dinner' purposes....
and not just because it is illegal. You never know what your colleague
has been doing witht he microwave...
>I'm not sure that anybody actually studied it but it's a good idea to keep
>food away from the hazardous chemicals anyway. Recently I've taken up
>adding EtBr to the loading buffer instead of the gel - far less EtBr to
>worry about (in the running buffer etc.), lower background and you can boil
>the agarose as much as you want.
Different froms of DNA, like circular vs. linear migrate differently
in the presence or absence of EtBr. My advice is to run agarose
gels without EtBr, and then stain them for 5' afterwards. IMH, this gives the
nicest results.
clemens
Clemens Suter-Crazzolara
On 25 Apr 1998 17:35:59 +0100, Wolfgang Schechinger said:
>Hi Hiranya!
>You just boil your gel after taking the photo? - great! What
>about an increasing background by DNA? is there any?
>Wolfgang
>> I do it all the time and EtBr stays put. This way, I've been able to
>> reuse agarose at least 5 times without having to add significant
>> amts of EtBr. The savings are substantial too. This, of course, is
>> only for analytical purposes.
I would be careful about this method. after a while the resolution
becomes quite bad, because the agarose starts breaking down.
It is probably better to buy a cheaper agarose for quicky gels.
clemens
Martin Gerken
>food away from the hazardous chemicals anyway. Recently I've taken up
>adding EtBr to the loading buffer instead of the gel - far less EtBr to
>worry about (in the running buffer etc.), lower background and you can boil
>the agarose as much as you want.
Unfortunately, EtBr moves in the opposite direction of the DNA...
I guess, you have to use far more EtBr as if you put the EtBr directly
into the gel.
cu, Martin
Vladimir Svetlov
In article <6i437i$2...@sun0.urz.uni-heidelberg.de>,
un6...@genius.embnet.dkfz-heidelberg.de wrote:
> >A good practice is to use a separate microwave for this and reheating food.
>
> generally, I wouldn't use ANY laboratory instrument for 'dinner' purposes....
> and not just because it is illegal. You never know what your colleague
> has been doing witht he microwave...
"Generally" is a good word. I would not think - untill I saw it done - that
people would use a hot plate/stirrer to make noodles or heat up stripping
solutions in the same microwave others use to warm up lunches... Unless
there are two microwaves in the lab - one of them in the lunch room -
people would cook agarose and sandwiches in the same one. Ye know, after i
found a frozen Tyson chicken in the -80 C freezer next to frozen calf
thymus, yeast, E. coli and Salmolnella cell masses I ain't taking any
chances with people's common sense.
> >I'm not sure that anybody actually studied it but it's a good idea to keep
> >food away from the hazardous chemicals anyway. Recently I've taken up
> >adding EtBr to the loading buffer instead of the gel - far less EtBr to
> >worry about (in the running buffer etc.), lower background and you can boil
> >the agarose as much as you want.
>
> Different froms of DNA, like circular vs. linear migrate differently
> in the presence or absence of EtBr. My advice is to run agarose
> gels without EtBr, and then stain them for 5' afterwards. IMH, this gives the
> nicest results.
Depending on the thickness of the gel staining and destaining may take up
far longer than 5'. And of course I was not talking about application
(absent from my own cloning work) that require separation of different
helical forms of DNA. So as an alternative to having lotsa EtBr in the gel
(and eventually in the running buffer or staining/destaining solutions)
adding it in smaller amounts to the DNA is not so bad.
Regards,
V.
Vladimir Svetlov
In article <3545f045...@news.uni-koeln.de>,
a288...@smail.uni-koeln.de (Martin Gerken) wrote:
>> Recently I've taken up
> >adding EtBr to the loading buffer instead of the gel - far less EtBr to
> >worry about (in the running buffer etc.), lower background and you can boil
> >the agarose as much as you want.
>
> Unfortunately, EtBr moves in the opposite direction of the DNA...
> I guess, you have to use far more EtBr as if you put the EtBr directly
> into the gel.
Unbound EtBr, you mean? But that's good. Let it. Since it's travelling to
the catode from the well (and not from across the entire gel) it's not
gonna affect the brightness of the field at all. Normally you'd have to
wait a while before the bottom part of your gel is cleared from the EtBr.
On the other hand, bound to DNA EtBr travels with it to the anode - these
intercalating dyes don't dissociate that easy - so that yer DNA remains
nice and shiny in the UV. So I don't think that this metaphysical argument
really stands up.
Stephan
In article <svetlov-ya0240800...@news.doit.wisc.edu>,
sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
> In article <3545f045...@news.uni-koeln.de>,
> a288...@smail.uni-koeln.de (Martin Gerken) wrote:
>
> >> Recently I've taken up
> > >adding EtBr to the loading buffer instead of the gel - far less EtBr to
> > >worry about (in the running buffer etc.), lower background and you can boil
> > >the agarose as much as you want.
> >
> > Unfortunately, EtBr moves in the opposite direction of the DNA...
> > I guess, you have to use far more EtBr as if you put the EtBr directly
> > into the gel.
>
> Unbound EtBr, you mean? But that's good. Let it. Since it's travelling to
> the catode from the well (and not from across the entire gel) it's not
> gonna affect the brightness of the field at all. Normally you'd have to
> wait a while before the bottom part of your gel is cleared from the EtBr.
> On the other hand, bound to DNA EtBr travels with it to the anode - these
> intercalating dyes don't dissociate that easy - so that yer DNA remains
> nice and shiny in the UV. So I don't think that this metaphysical argument
> really stands up.
Mmmm... I wouldn't be so sure about the dissociation. EtBr DOES dissociate
from the DNA, and quite well I would say (have you ever done EtBr-CsCl
extractions?). If the concentration of EtBr is constant in the gel and in
the buffer, an equilibrium is reached. The dissociated molecules will move
toward the cathode, while the DNA will move toward the anode. If there's
no EtBr to replace the one that has just dissociated and moved towards the
cathode, your bands will appear more and more faint, eventually until you
can't see them anymore.
There's no need of methaphysics to understand that.
Vladimir Svetlov
In article <REPLACE-2904...@macbb2212b.unil.ch>,
REP...@lbm.unil.ch (Stephan) wrote:
> > Unbound EtBr, you mean? But that's good. Let it. Since it's travelling to
> > the catode from the well (and not from across the entire gel) it's not
> > gonna affect the brightness of the field at all. Normally you'd have to
> > wait a while before the bottom part of your gel is cleared from the EtBr.
> > On the other hand, bound to DNA EtBr travels with it to the anode - these
> > intercalating dyes don't dissociate that easy - so that yer DNA remains
> > nice and shiny in the UV. So I don't think that this metaphysical argument
> > really stands up.
>
> Mmmm... I wouldn't be so sure about the dissociation.
Well, if you would be so kind as to provide the dissociation constant and
the hal-life for EtBr-DNA complex in TBE, we could actually leave the
metaphysical ground. Oh, shit, I forgot "Mmmm".
> EtBr DOES dissociate
> from the DNA, and quite well I would say (have you ever done EtBr-CsCl
> extractions?).
Never in my life. I did EtBr extractions by n-butanol or using Dowex column
from CsCl solutions. Neither extracting agent is a component of my
electrophoretic protocol. Mmmm.
> If the concentration of EtBr is constant in the gel and in
> the buffer, an equilibrium is reached.
Even in recirculating buffer there always be a gradient pattern of EtBr in
the electric field and ... Stop, even better - please right out a kinetic
analysis of EtBr-DNA dissociation for a compact band of a finite volume
instead of infinitely dilute and momentarily equilibrating solution.
>The dissociated molecules will move
> toward the cathode, while the DNA will move toward the anode. If there's
> no EtBr to replace the one that has just dissociated and moved towards the
> cathode, your bands will appear more and more faint, eventually until you
> can't see them anymore.
Same thing would "eventually" happen in the gel with EtBr added to it,
would it not? Are you going to such a great length to justify the primal
urge to flood everything including the running buffer with EtBr?
It's too bad that Polya's work on plausible reasoning is not a required
reading for biology students. Apart from being a truism, this deliberation
sadly lacks any indication to what condition one have to establish for the
gel run for this dissociation to actually become a problem for nucleic
acids detection with UV.
> There's no need of methaphysics to understand that.
Tha'sss right, my lad. What needed though is some knowledge of kinetics.
Unless you are willing to entertain my questions above I think we can cease
this pointless discussion of why things should not work the way they do.
Frankly speaking, if the need comes for that kinda mental exercise I'd
rather take it up with Zeno (no offense, man).
Have fun,
Stephan
In article <svetlov-ya0240800...@news.doit.wisc.edu>,
sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
> Well, if you would be so kind as to provide the dissociation constant and
> the hal-life for EtBr-DNA complex in TBE, we could actually leave the
> metaphysical ground. Oh, shit, I forgot "Mmmm".
Why are you beeing so aggressive?
You can find the physico-chemical characterization of the NA-EtBr
interactions in:
LePecq JB. Paoletti C.
J. Mol. Biol. 27(1):87-106, 1967.
For a review, see:
LePecq et al.
Meth. Bioch. Anal., 20:41-86, 1971.
For the rest, I won't answer your provocative and nearly insulting posting.
You seem to have completely missed the point. I was interested to know a
little more about a method that I had never used before and I said that I
was skeptical about a general application of the method. I have more years
of experience running agarose gels than you are probably assuming. Don't
assume on me, you don't know me.
Have a nice day, and please don't dump your aggressivity so easely.
Stephan
Chris Boyd
Stephan (REP...@lbm.unil.ch) wrote:
: In article <svetlov-ya0240800...@news.doit.wisc.edu>,
: sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
: > In article <3545f045...@news.uni-koeln.de>,
: > a288...@smail.uni-koeln.de (Martin Gerken) wrote:
: >
: > >> Recently I've taken up
: > > >adding EtBr to the loading buffer instead of the gel - far less EtBr to
: > > >worry about (in the running buffer etc.), lower background and you can boil
: > > >the agarose as much as you want.
: > >
: > > Unfortunately, EtBr moves in the opposite direction of the DNA...
: > > I guess, you have to use far more EtBr as if you put the EtBr directly
: > > into the gel.
: >
: > Unbound EtBr, you mean? But that's good. Let it. Since it's travelling to
: > the catode from the well (and not from across the entire gel) it's not
: > gonna affect the brightness of the field at all. Normally you'd have to
: > wait a while before the bottom part of your gel is cleared from the EtBr.
: > On the other hand, bound to DNA EtBr travels with it to the anode - these
: > intercalating dyes don't dissociate that easy - so that yer DNA remains
: > nice and shiny in the UV. So I don't think that this metaphysical argument
: > really stands up.
: Mmmm... I wouldn't be so sure about the dissociation. EtBr DOES dissociate
: from the DNA, and quite well I would say (have you ever done EtBr-CsCl
: extractions?). If the concentration of EtBr is constant in the gel and in
: the buffer, an equilibrium is reached. The dissociated molecules will move
: toward the cathode, while the DNA will move toward the anode. If there's
: no EtBr to replace the one that has just dissociated and moved towards the
: cathode, your bands will appear more and more faint, eventually until you
: can't see them anymore.
I can personally vouch for the destaining of bands in an agarose gel.
On several occasions, I've run pre-stained gels for a long time and
found that the lower MW bands have become invisible. (That's why you
always, always need a size marker on a gel to see if this has
occurred.) The missing bands reappear if the gel is stained after
running. In general, though, all you see is reduced intensity of the
lower MW bands due to partial dissociation as the EtBr front zooms
towards the cathode. That's molecular biology, not metaphysics.
Best wishes,
--
Chris Boyd | from, but not \ MRC Human Genetics Unit,
chr...@hgu.mrc.ac.uk | on behalf of / Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \ Edinburgh, EH4 2XU, SCOTLAND
Vladimir Svetlov
In article <REPLACE-3004...@macbb2212b.unil.ch>,
REP...@lbm.unil.ch (Stephan) wrote:
> In article <svetlov-ya0240800...@news.doit.wisc.edu>,
> sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
>
> > Well, if you would be so kind as to provide the dissociation constant and
> > the hal-life for EtBr-DNA complex in TBE, we could actually leave the
> > metaphysical ground. Oh, shit, I forgot "Mmmm".
>
> Why are you beeing so aggressive?
You have not seen me being agressive. As for explanation of my percepted
agressiveness - I hate this condensending "Mmmm" preceeding a rather
pointless and tangential truism that constituted your post. I don't
consider contrivancies as "Have you ever done EtBr-CsCl extractions" a
serious argument, not untill you demonstrate how organic extraction of EtBr
is similar to gel-electrophoresis. Have you try to extract EtBr with TBE?
> You can find the physico-chemical characterization of the NA-EtBr
> interactions in:
>
> LePecq JB. Paoletti C.
> J. Mol. Biol. 27(1):87-106, 1967.
>
> For a review, see:
>
> LePecq et al.
> Meth. Bioch. Anal., 20:41-86, 1971.
>
> For the rest, I won't answer your provocative and nearly insulting posting.
Did I try to provoke you to actually do some kinetics calculation? Oh, I'm
sooo sorry. It won't happen again. But it would be nice if you try to
support your hand-waving (I use this term in lieu of "metaphysics" which is
too long of a word for this debate) with some calculations.
> You seem to have completely missed the point.
There were no point to miss in your post. In private I already expressed my
insincere surprise that such a trivial matter as boiling gels attracts such
a great attention on this board, whereas many a topics of far greater
interest lie forgotten or overlooked. The point that you've missed is that
it's a very simple matter - it does not really matter where DNA binds EtBr,
it stays bound long enough and in amount sufficient to enable one to
visualize DNA fragments of various sizes during routine electrophoretic
separations. It is but one of the many way people use to stain DNA and
everyone is welcome to find one that fits his/her idiosyncrasy of the day.
> I was interested to know a
> little more about a method that I had never used before and I said that I
> was skeptical about a general application of the method.
You use the word method too lightly. This is but a variegation on the
common theme and it works well within the limits of applicability which are
obvious. They must have forgotten to tell you that just being sceptical is
no longer enough for a good grade.
> I have more years
> of experience running agarose gels than you are probably assuming. Don't
> assume on me, you don't know me.
Don't forget to put on your resume.
Vladimir Svetlov
In article <6i9qe2$2...@scotsman.ed.ac.uk>, chr...@hgu.mrc.ac.uk (Chris
Boyd) wrote:
> I can personally vouch for the destaining of bands in an agarose gel.
> On several occasions, I've run pre-stained gels for a long time and
> found that the lower MW bands have become invisible. (That's why you
> always, always need a size marker on a gel to see if this has
> occurred.) The missing bands reappear if the gel is stained after
> running. In general, though, all you see is reduced intensity of the
> lower MW bands due to partial dissociation as the EtBr front zooms
> towards the cathode. That's molecular biology, not metaphysics.
I don't see where biology comes into that, but hell with it. Metaphysics
and Zeno were invoked here for the purpose of pointing out that a
needlessly "theoretical" discussion of a very simple technical "tip" had
swept this board. By far that's the longest thread I've seen here and what
ridiculously trivial the problem is...
As far as your "voucher" is concerned - indeed, bands do destain unless
EtBr is present in the running buffer in addition to that in the gel and
buffer is recirculated. And, mind you, I never argued otherwise. However,
even when EtBr front travels past the DNA bands their UV visibility does
not diminish momentarily, does it?. For longer gels it may be necessary to
stain them afterwards to visualize the bands either way you run them, for
many runs - like minipreps or preparative gels - it is not. In case of
preparative gels one even might find that less EtBr serves'im better than
an excess of it. So every technique has it's own limits and advantages,
including running gels with no EtBr and staining it afterwards. This is a
false antagonism prompted by apparently adolescent desire to find the
universal method for everything. I've been a witness to a hot debate once
whether DNA should be stained rapidly in high conc. of EtBr or slowly in
low. Care to take a vote on this one?
To (hopefully) wrap it up - it's obvious why adding EtBr to DNA works
and it's obvious when it won't work; any takers can try it and if they find
that it does not suit their needs they can discontinue it's use immediately
and absolutely free of charge.
Regards,
Chris Boyd
Vladimir Svetlov (sve...@oncology.wisc.edu) wrote:
: In article <6i9qe2$2...@scotsman.ed.ac.uk>, chr...@hgu.mrc.ac.uk (Chris
: Boyd) wrote:
: > I can personally vouch for the destaining of bands in an agarose gel.
: > On several occasions, I've run pre-stained gels for a long time and
: > found that the lower MW bands have become invisible. (That's why you
: > always, always need a size marker on a gel to see if this has
: > occurred.) The missing bands reappear if the gel is stained after
: > running. In general, though, all you see is reduced intensity of the
: > lower MW bands due to partial dissociation as the EtBr front zooms
: > towards the cathode. That's molecular biology, not metaphysics.
: I don't see where biology comes into that, but hell with it.
If you'd waited until a little of that blood drained from your eyes,
you might have seen the word `molecular' in front of `biology'.
: Metaphysics
: and Zeno were invoked here for the purpose of pointing out that a
: needlessly "theoretical" discussion of a very simple technical "tip" had
: swept this board. By far that's the longest thread I've seen here and what
: ridiculously trivial the problem is...
: As far as your "voucher" is concerned - indeed, bands do destain unless
: EtBr is present in the running buffer in addition to that in the gel and
: buffer is recirculated. And, mind you, I never argued otherwise. However,
: even when EtBr front travels past the DNA bands their UV visibility does
: not diminish momentarily, does it?. For longer gels it may be necessary to
: stain them afterwards to visualize the bands either way you run them, for
: many runs - like minipreps or preparative gels - it is not. In case of
: preparative gels one even might find that less EtBr serves'im better than
: an excess of it.
So we're agreed. Why the follow-up?
: So every technique has it's own limits and advantages,
: including running gels with no EtBr and staining it afterwards. This is a
: false antagonism prompted by apparently adolescent desire to find the
: universal method for everything.
You won't get far in science by publicly characterising the motives of
people who argue with you as `adolescent'.
: I've been a witness to a hot debate once
: whether DNA should be stained rapidly in high conc. of EtBr or slowly in
: low. Care to take a vote on this one?
: To (hopefully) wrap it up - it's obvious why adding EtBr to DNA works
: and it's obvious when it won't work; any takers can try it and if they find
: that it does not suit their needs they can discontinue it's use immediately
: and absolutely free of charge.
It's far from obvious. That's why there's a debate. It's the purpose
of the ng to discuss such matters when there's a difference of opinion.
I agree that too many of the opinions expressed are not backed up by
experimental data, but there's nothing to be gained (as you've seen) by
using inflammatory language.
Stephan
In article <svetlov-ya0240800...@news.doit.wisc.edu>,
sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
> To (hopefully) wrap it up - it's obvious why adding EtBr to DNA works
> and it's obvious when it won't work; any takers can try it and if they find
> that it does not suit their needs they can discontinue it's use immediately
> and absolutely free of charge.
> Regards,
> V.
I have to admit that you finally close that "EtBr to DNA" pseudodiscussion
in a quite elegant manner. Or so I hope, too. Congratulations.
Originally my question was: can I apply that "tip" to my daily experiments?
Finally I got the answer: no I can't. Unless I plan to do some easy
minipreps or some easy preparative gels (something that doesn't happen
everyday). Of course, I could have answered that question experimentylly
by myself. But I know now that it would have been a waste of time. If not
of energy and money. What would be the point otherwise to have a forum
were the topics are supposed to be "methods and reagents in molecular
biology"?
No "tip" is trivial to me, even if I have been using it for years. Even
less would I pretend that my eventual "tips" should be trivial to others.
Anyway, thank you very much for your information.
I apologize for maybe not having made my question clearer from the beginning.
Have all a nice day,
Stephan
Hung-yiu Ho
Even a trivial thing may cause problem. Even though the experienced phD
like you may thoroughly understand what happens during DNA agarose
electrophoresis, some newcomers or greenhands may not. In addition, some
people are really interested in kinetics and physics involved in such
process. Nobody have the right to decide what is actually important and
what should be discussed in this newsgroup. Science means freedom of
knowledge. I can recall that a paper describing the DNA gel
electrophoresis was published in Nature a couple of years ago. Something
that appeared to be insignicant, like small bug C. elegan, will turns to
be big things years later.
On Thu, 30 Apr 1998, Vladimir Svetlov wrote:
> In article <REPLACE-3004...@macbb2212b.unil.ch>,
> REP...@lbm.unil.ch (Stephan) wrote:
>
> > In article <svetlov-ya0240800...@news.doit.wisc.edu>,
> > sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
> >
Clemens Suter-Crazzolara
On Wed, 29 Apr 1998 10:04:28 -0600, Vladimir Svetlov said:
>In article <REPLACE-2904...@macbb2212b.unil.ch>,
>REP...@lbm.unil.ch (Stephan) wrote:
> >The dissociated molecules will move
> > toward the cathode, while the DNA will move toward the anode. If there's
> > no EtBr to replace the one that has just dissociated and moved towards the
> > cathode, your bands will appear more and more faint, eventually until you
> > can't see them anymore.
>Same thing would "eventually" happen in the gel with EtBr added to it,
>would it not? Are you going to such a great length to justify the primal
>urge to flood everything including the running buffer with EtBr?
that's why I say: run the gel, THEN stain 5 minutes in EtBr !
Saves on EtBr, AND your DNA will run acc. to its properties, NOT those of
DNA + EtBr ! And your geltanks won't get contaminated with EtBr.
But well, why am I standing here shouting in the desert.
Could we have a vote on the nest method ?
>It's too bad that Polya's work on plausible reasoning is not a required
>reading for biology students. Apart from being a truism, this deliberation
>sadly lacks any indication to what condition one have to establish for the
>gel run for this dissociation to actually become a problem for nucleic
>acids detection with UV.
V., it IS a problem. I've tried all methods. Yours is the least superior, and
the DNA does loose its EtBr the fastest, compared with all other methods. Faint bands
are no longer visible after prolonged runs.
>Tha'sss right, my lad. What needed though is some knowledge of kinetics.
Nah, common sense, and the experiments to support them. I like your sarcasm though.
clemens
Clemens Suter-Crazzolara
On Thu, 30 Apr 1998 12:13:08 -0600, Vladimir Svetlov said:
>> I can personally vouch for the destaining of bands in an agarose gel.
>> On several occasions, I've run pre-stained gels for a long time and
>> found that the lower MW bands have become invisible. (That's why you
...
>I don't see where biology comes into that, but hell with it. Metaphysics
>and Zeno were invoked here for the purpose of pointing out that a
>needlessly "theoretical" discussion of a very simple technical "tip" had
>swept this board. By far that's the longest thread I've seen here and what
>ridiculously trivial the problem is...
Well, as long as you keep giving the wrong advice to potential newbies
which may read your mails, don't be surprised if people jump on
you. Those of us that have been in the field for some time, know that
running agarose gels is not a trivial thing !
Secondly, it appears that it is YOU who keeps this tread going and going....
Mmmmm, with this message it could also be ME admittedly.
clemens
Vladimir Svetlov
In article <Stephan.Heeb-2...@remote-acc-17.unil.ch>,
Stepha...@Lbm.unil.ch (Stephan Heeb) wrote:
> Okay, okay! But please stay cool! You overheated, then I turned the power
> down! I have stoped using that "Mmmm" in my posts, did you notice? Now
> could you stop having such a deffensive attitude? Nobody here so far has
> been prosecuting you!
How does the prosecutor plead?
> > I don't
> > consider contrivancies as "Have you ever done EtBr-CsCl extractions" a
> > serious argument, not untill you demonstrate how organic extraction of EtBr
> > is similar to gel-electrophoresis. Have you try to extract EtBr with TBE?
>
> No I haven't. But I have noticed for several years (and others have
> noticed that, too) that DNA-EtBr bands in agarose gels become faint
And everybody else did. Common place, truism, trivial.
> Are you going to pretend that this is not a valid argument? Can we all
> make in our labs a simple experiment to confirm your hypotesis that EtBr
> doesn't dissociate from DNA in agarose gels?
I did not form such a hypothesis. I stated that this dissociation is slow
enough to enable one to use the modification in question.
> > Did I try to provoke you to actually do some kinetics calculation? Oh, I'm
> > sooo sorry. It won't happen again. But it would be nice if you try to
> > support your hand-waving (I use this term in lieu of "metaphysics" which is
> > too long of a word for this debate) with some calculations.
>
> I will do the calculations that you want if you prove me that I'm wrong. I
> pretend that "EtBr dissociates from DNA in agarose gels", and I can
> experimentally prove it.
It is expected from the "theoretician" to provide a solid proof for his
assertion at the time when the thesis is submitted for public scrutiny. If
it did not concern you personally even you would agree that after a proof
of your wrong no one would be much interested in your original
calculations. Since you neither have them nor capacity to their production,
we might as well drop this subject altogether.
> Now, prove us that this is not true, and I'll
> calculate anything you want. I will even convert myself into a metaphysist
> if you prove that our science is wrong.
"Our science"? There are more?!
> As I say every year to the students that I'm in charge of: "there are no
> stupid questions, only stupid answers".
I'm sure they get enough of those.
> This agarose gel topic may be too
> trivial to you. Okay. Let it. But then, why are you loosing your time
> arguing with me?
Bad temper.
> > The point that you've missed is that
> > it's a very simple matter - it does not really matter where DNA binds EtBr,
> > it stays bound long enough and in amount sufficient to enable one to
> > visualize DNA fragments of various sizes during routine electrophoretic
> > separations. It is but one of the many way people use to stain DNA and
> > everyone is welcome to find one that fits his/her idiosyncrasy of the day.
>
> I fully agree with you.
You can not fully agree with person you just argued with.
Skipped a bit.
> Anyway, I'm sure that you must be good at something. If you are good
> enough to prove us that EtBr doesn't dissociate from DNA in agarose gels,
> I promise to help you to submit a paper to a good journal. ;^)
If the Medline gives us both justice, you need this help more than I do.
Well, I hope for their sake that the people on alt.chinchilla got the best
of your wits and knowledge...
Regards,
Vladimir Svetlov
In article <6ic66u$q...@scotsman.ed.ac.uk>, chr...@hgu.mrc.ac.uk (Chris
Boyd) wrote:
> If you'd waited until a little of that blood drained from your eyes,
> you might have seen the word `molecular' in front of `biology'.
Now, that's but a glimpse of the English wit... Or is it Scottish? Still
better than the rest. Bravo! And a double snap. When I went to school, the
term molecular biology was applied to study of biological systems on the
molecular level rather than a compendium of techniques used in the process.
That's an explanation for the minor snap.
> So we're agreed. Why the follow-up?
Me asked first, my preciousss, yesss, me asked first. Why sooo many people
charged to the defense of the thesis no one (not usss, my precious, not
usss) had assailed in the first place? Because they are nasty and they type
faster then they read, that'sss why.
> : So every technique has it's own limits and advantages,
> : including running gels with no EtBr and staining it afterwards. This is a
> : false antagonism prompted by apparently adolescent desire to find the
> : universal method for everything.
>
> You won't get far in science by publicly characterising the motives of
> people who argue with you as `adolescent'.
That's right. Must find a nastier word, must find it...
> : I've been a witness to a hot debate once
> : whether DNA should be stained rapidly in high conc. of EtBr or slowly in
> : low. Care to take a vote on this one?
>
> : To (hopefully) wrap it up - it's obvious why adding EtBr to DNA works
> : and it's obvious when it won't work; any takers can try it and if they find
> : that it does not suit their needs they can discontinue it's use immediately
> : and absolutely free of charge.
>
> It's far from obvious. That's why there's a debate. It's the purpose
> of the ng to discuss such matters when there's a difference of opinion.
> I agree that too many of the opinions expressed are not backed up by
> experimental data, but there's nothing to be gained (as you've seen) by
> using inflammatory language.
What you call nothing?! The fact that for many participants of this thread
it was but their first contribution to this board?! That we came up with
the longest and pointelessnest thread on the board?! You called that
nothing?!
Vladimir Svetlov
In article <6ikcqu$n...@sun0.urz.uni-heidelberg.de>,
un6...@genius.embnt.dkfz-heidelberg (Clemens Suter-Crazzolara) wrote:
> >Same thing would "eventually" happen in the gel with EtBr added to it,
> >would it not? Are you going to such a great length to justify the primal
> >urge to flood everything including the running buffer with EtBr?
>
> that's why I say: run the gel, THEN stain 5 minutes in EtBr !
> Saves on EtBr, AND your DNA will run acc. to its properties, NOT those of
> DNA + EtBr ! And your geltanks won't get contaminated with EtBr.
> But well, why am I standing here shouting in the desert.
> Could we have a vote on the nest method ?
This is too idiosyncratic to take a vote. Yes, I did exactly that for the
DNA fingerprinting gels, where separation takes place over several hours
worth of running time and no EtBr would survive that. For the gels run over
a couple of hours this is not absolutely neccesary and for most preparative
applications or PCR-based techniques addition of EtBr to the DNA more than
adequate.
There was a student in me old lab that would on principle run gels at 10
V/sm for 15 minutes when she was screening the mini's for the insert and
was satisfied with the bands separated from the backbone by a mm or two -
do you think she could wait for staining and destaining to take place?!!!
Life is too short...
> V., it IS a problem. I've tried all methods. Yours is the least superior, and
> the DNA does loose its EtBr the fastest, compared with all other methods.
Faint bands
> are no longer visible after prolonged runs.
I never claimed it was superior - just that it is takes less EtBr and does
not require presence of EtBr in the agarose that's being boiled (the latter
goes for your favourite method), so that for situations when people are
more concerned with the contamination than finer points in gel running (for
routine or large-scale applications) it is a reasonable alternative. Now we
gone a long way from that starting question of agarose boiling, have we
not?
Stephan
Vladimir Svetlov wrote:
> Well, I hope for their sake that the people on alt.chinchilla got the best
> of your wits and knowledge...
Sheeesh! You *really* have a lot of time to waste! Yes, I have some
interest for chinchillas when not doing molecular biology. Where's the
problem?
Stephan
NB: I would have prefered to meet you in alt.fan.tolkien instead of here.
You're not so funny in this NG. But maybe we have already met in MUME? ;-)
Stephan
Vladimir Svetlov, the new bionet troll guy wrote:
> > It's far from obvious. That's why there's a debate. It's the purpose
> > of the ng to discuss such matters when there's a difference of opinion.
> > I agree that too many of the opinions expressed are not backed up by
> > experimental data, but there's nothing to be gained (as you've seen) by
> > using inflammatory language.
>
> What you call nothing?! The fact that for many participants of this thread
> it was but their first contribution to this board?! That we came up with
> the longest and pointelessnest thread on the board?! You called that
> nothing?!
Gosh! The Internet trolls have evolved into a superior degree!
Now, should we have to expect the whole bionet.* NG to be invaded by such
rubbish threads? I this was the case, I would definitely deduce that
there's too much people out there that has nothing else to do...
Well, since I'm not against evolution (and I guess none of you are), let's
face it and see how long this kind of new "stupid thread producing
species" will survive.
Stephan
Vladimir Svetlov
In article <S.H-050598...@macbb2212b.unil.ch>, S...@lbm.unil.ch
(Stephan) wrote:
> Vladimir Svetlov wrote:
>
>
> > Well, I hope for their sake that the people on alt.chinchilla got the best
> > of your wits and knowledge...
>
> Sheeesh! You *really* have a lot of time to waste! Yes, I have some
> interest for chinchillas when not doing molecular biology. Where's the
> problem?
It does not take much time to check a profile of a Usenet contributor,
really. Contrary to your belief I don't assume much, rather I prefer to
check my speculations and/or impressions against the data before I make
them public. I got an impression that you would not be much of a
contributor to scientific board (indeed) and of course I'd like to find
some whimsical board that you do contribute a great deal on a regular
basis. Alt.chinchilla seemed like a perfect choice. Ad hominem attack, the
weapon of choice on talk.origins.
Now to the problem. It's actually two-fold. One is that even sarcasm goes
pretty much over your head - that's why I would not respond to your next
posting. The other is that from your postings I saw already and the pattern
of your contribution I'd conclude that the you "do" (intended pun)
molecular biology in that little time left from this fascination with
chinchillas, rather than the other way around. There is nothing to be
ashamed of - a lot of folks out there don't really like the job they are
paid for and find an outlet for their creativity and compassion in flower
arrangements, haiku or teaching trigonometry to small and fuzzy critters.
I'm actually proud of you, my friend, since admission that you have a
problem is the first step leading to recovery. Or conviction. You see, I'm
also a HSUS and ASPCA member, so I'll be watching you and those
chinchillas.
Dr. Bob
>In article <S.H-050598...@macbb2212b.unil.ch>, S...@lbm.unil.ch
>(Stephan) wrote:
>> Vladimir Svetlov wrote:
>> > Well, I hope for their sake that the people on alt.chinchilla got the best
>> > of your wits and knowledge...
>> Sheeesh! You *really* have a lot of time to waste! Yes, I have some
>> interest for chinchillas when not doing molecular biology. Where's the
>> problem?
>It does not take much time to check a profile of a Usenet contributor,
>really.
>Vladimir Svetlov, Ph. D.
Vladimir Svetlov: are you spying on us ?
clemens
Cornelius Krasel
http://www.dejanews.com/ will allow everybody to create a user profile
of a certain mail address. Not much spying involved, I suppose.
--Cornelius.
--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: pha...@rzbox.uni-wuerzburg.de SP4 */
/* "Science is the game we play with God to find out what His rules are." */
Vladimir Svetlov
In article <6iq1ft$6...@sun0.urz.uni-heidelberg.de>,
un6...@genius.embnet.dkfz-heidelberg.de (Dr. Bob) wrote:
> >It does not take much time to check a profile of a Usenet contributor,
> >really.
>
> >Vladimir Svetlov, Ph. D.
>
>
> Vladimir Svetlov: are you spying on us ?
>
>
> clemens
My supervisor in KGB (FSB) told me to deny it, so of course not. No, not at
all, no, no and no again. I never! Absolutely not! Me?!
Actually, yes. How else are you going to find a material for a good ad
hominem attack? And web gives a plenty opportunities for that, because
people are so vane in the making of their web pages, and so oblivious of
the fact that the Net has a long memory of their submissions to the
alt.pictures.tasteless. I learned my moves on t.o. and as soon as we leave
the scientific territory and go at each other personally, I do not leave a
stone unturned untill I find something funny. On my own time, of course.
Late at night developing the westerns. They don't call me Vlad the Impaler
for nothing, you know.
Stephan
Vladimir Svetlov wrote:
> > > Well, I hope for their sake that the people on alt.chinchilla got the best
> > > of your wits and knowledge...
> >
> > Sheeesh! You *really* have a lot of time to waste! Yes, I have some
> > interest for chinchillas when not doing molecular biology. Where's the
> > problem?
>
> It does not take much time to check a profile of a Usenet contributor,
> really.
If it doesn't take time to you it's surely because you have an insane
interest at sneaking other's lifes and you have become an expert at that.
> Contrary to your belief I don't assume much, rather I prefer to
> check my speculations and/or impressions against the data before I make
> them public.
Ha! Ha! Ha! It's that a joke? See below...
> I got an impression that you would not be much of a
> contributor to scientific board (indeed) and of course I'd like to find
> some whimsical board that you do contribute a great deal on a regular
> basis. Alt.chinchilla seemed like a perfect choice.
Do I contribute to alt.chinchilla in a "regular" basis?!? Please check
again the speculative profile that you are assuming that I have with no
real basis other than what anybody can find on the Web. Not one post in
the last year, that makes me a "regular" contributor to that NG? Hum!
Instead, did you know that I posted something two weeks ago on alt.ketchup
and that now one of my favorites newsgroups is rec.food.cooking? I can't
believe that you're beeing serious.
> The other is that from your postings I saw already and the pattern
> of your contribution I'd conclude that the you "do" (intended pun)
> molecular biology in that little time left from this fascination with
> chinchillas, rather than the other way around.
If you're so clever, check out WHEN was the last time that I posted
something to alt.chinchillas. And even if I had posted something
yesterday, you would still be wildly speculating without any knowledge
about how I spend my time. I don't like, as you do, to speculate about
other's lifes, but here the one that really needs help is you: don't sneak
in my private life as nobody else here is sneaking in yours!
> There is nothing to be
> ashamed of - a lot of folks out there don't really like the job they are
> paid for and find an outlet for their creativity and compassion in flower
> arrangements, haiku or teaching trigonometry to small and fuzzy critters.
Or spying other's lifes...
> I'm actually proud of you, my friend, since admission that you have a
> problem is the first step leading to recovery. Or conviction. You see, I'm
> also a HSUS and ASPCA member, so I'll be watching you and those
> chinchillas.
Great! Want to see pictures of my pets? I have plenty of them, but not
(yet) a homepage... ;-) Want to know how I build a huge but not less
inexpensive cage? I would prefer that kind of information exchange between
us instead of having sterile talks about our professional and
non-professional skills.
Regards,
Stephan
BTW, you didn't answer my question: could we have met in MUME?
Vladimir Svetlov
In article <S.H-070598...@macbb2212b.unil.ch>, S...@lbm.unil.ch
(Stephan) wrote:
Your five pounds worth of arguing are long gone, my lad. Anyhow...
> Do I contribute to alt.chinchilla in a "regular" basis?!?
More regular than to methds-reagnts.
> Instead, did you know that I posted something two weeks ago on alt.ketchup
> and that now one of my favorites newsgroups is rec.food.cooking?
Don't like ketchup. Here in States food is not a joking matter and
referring to it in such a context would be next to a sacrilege. Now, if
I've seen your photo that proved that all that food frenzy impacted heavily
on yer figure, than I just might have wanna use that...
>I can't
> believe that you're beeing serious.
I'm not.
Cheers,
Stephan
In article <svetlov-ya0240800...@news.doit.wisc.edu>,
sve...@oncology.wisc.edu (Vladimir Svetlov) wrote:
> > Do I contribute to alt.chinchilla in a "regular" basis?!?
>
> More regular than to methds-reagnts.
Quantity is not equal to quality. I try not to open my mouth when
unnecessary in this NG (excepted for this thread, of course). Note that
this is not necessarily true for other NGs. I sometimes like to play with
some especially irritable netters (of course, I use pseudonyms and fake
e-mail addresses when doing that, I don't like to be mailbombed or traced
back with DejaNews ;-)
> > Instead, did you know that I posted something two weeks ago on alt.ketchup
> > and that now one of my favorites newsgroups is rec.food.cooking?
>
> Don't like ketchup.
I don't like it either. That's why I do monitor <alt.ketchup>... ;-)
> Here in States food is not a joking matter and
> referring to it in such a context would be next to a sacrilege.
ROTFLMAO!
> Now, if
> I've seen your photo that proved that all that food frenzy impacted heavily
> on yer figure, than I just might have wanna use that...
I don't think ketchup is a relevant factor determining the human body
shape, but I have no data to prove my point. There are a few studies that
have been done on ketchup (see Medline) but they only deal such subjects
as the tooth wear in teenagers or the viscoelastic properties of horse
blood...
> I can't believe that you're beeing serious.
>
> I'm not.
You are seriously unserious.
Stephan