Fw: EtBr absorbance

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Mar 15 11:52:32 EST 2001


Yes.  My stock is 10 mg/ml and I use only 1 ul per 100 ml gel.  For most
routine DNA analyses (e.g. restriction digest to confirm identity of a
clone, etc.) I put the ethidium only in the gel.  Granted the ethidium will
run out of the gel, but most people look at their gel without running it
more than halfway anyway.  Besides, this is only a problem for faint bands.
If it is a concern, EtBr can be added to the running buffer too (at 0.1
ug/ml), but I don't really find I need to do this for most DNA gels I run.
I do put EtBr in both the gel and the running buffer for RNA analysis
(again, at 0.1 ug/ml final) though.

I suspect the 1 ug/ml figure you mention is for staining after running a
gel.  I almost never do this.  I suppose that perhaps the waste issue isn't
all that big of a deal, since, with 10 ul of EtBr stock, I could have stain
directly in 20 minigels, OR I could make up a staining solution and stain
20 minigels.  I bet most people would throw out their staining solution
before that--- or spill it!  Plus, I like to be able to pull the gel out of
the tank anytime I want and have it ready to look at.

Also, although SOP is good, it's not always the best.  Remeber, some
protocols become "codified" without really being optimized (few people like
to optimize, and if it's working good enough, most people aren't going to
take the time...).  One example is formaldehyde gels.  Many of the
conventional formulations have much more formaldehyde than needed.
Although I don't have a reference, I'm pretty certain it's been found that
less formaldehyde actually gave superior results, and made running the gel
less nasty!

Mike

>Mike
>Do you stain your gels in 0.1ug EtBr per ml?  I think SOP is around 1ug per
>ml (that would mean 10ul as jakku suggested).
>IMHO if the person who asked the original question wants only to stain a
>gel, maybe they should do a trial and error type of thing.  You know . . .
>Start with 5ul of "stock" solution in 100ml water.  Stain for 15 - 30 min,
>rinse, and visualize.
>If bands on the ladder are not bright enough, re-stain the same gel with
>10ul of "stock" . . . there might be a little diffusion of the bands but it
>should still be OK to read.
>
>I got the impression that this person was trying to do something else
>though.  If you are still out there, why do you want to know the
>concentration of the EtBr Solution?
>D Bell
>
>----------
>> From: "Michael L. Sullivan" <mlsulliv at facstaff.wisc.edu>
>> To: methods at hgmp.mrc.ac.uk
>> Subject: Re: EtBr absorbance
>> Date: Thursday, March 15, 2001 6:39 AM
>>
>> If you use only 1ul of 10 mg/ml EtBr per 100 ml directly in the gel it
>> works fine to stain.  AND this generates much less waste.
>>
>> Mike
>>
>> >hi...
>> >   why do you want to know the concentration of ethidium bromide
>solution.
>> >If it is for staining DNA gels, I don't think you need to bother much
>about
>> >concentrations whether it be 10 or 20mg/ml.  Just addd a little of this
>to
>> >water or buffer and you should be able to stain it.  around 10/20ul to
>100
>> >ml of solution.
>> >    bye
>> >jayakumar
>> >
>>
>> Michael L. Sullivan, Ph.D
>>
>> U.S. Dairy Forage Research Center
>> 1925 Linden Drive West
>> Madison WI, 53706
>>
>> (608) 264-5144 Phone
>> (608) 264-5147 Fax
>>
>> ---
>>
>
>---


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

---




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