EtBr absorbance

R. Jayakumar jakku at mrna.tn.nic.in
Sat Mar 17 00:08:04 EST 2001


Dear Mike
    thank you so much for clearing up the air.  I just posted several of the
responses I get from various researchers here on prestaining gels.  I too
agree with you and personally I do not find much difference, except in the
case of destaining prestained gels, which is very difficult, in case the
addition is in excess.
   It is also useful to keep the stained agarose gel at 4-8C in the
refrigerator,  for a few hours before documenting it.  This improves  the
resolution a lot.  It also reduces the background  and improves faint bands.
I don't know the principle behind it.  But it really works.  YOu will be
surprised.
   bye
cheers
jayakumar

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R. JAYAKUMAR
CSIR- Senior Research Fellow
Dept. of Molecular Microbiology,
School of Biotechnology,
Madurai Kamaraj University,
Madurai - 625 021.
India
email: jakku at linuxfan.com
tel: +91-452-858471-374
office fax(india)= +91-452-859105
Efax(USA): (603)-688-4665
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"Life is a comedy for those who think and a tragedy for those who
feel" --Emerson
----- Original Message -----
From: "Michael L. Sullivan" <mlsulliv at facstaff.wisc.edu>
To: "R. Jayakumar" <jakku at linuxfan.com>
Sent: Thursday, March 15, 2001 10:42 PM
Subject: Re: EtBr absorbance


> >Prestained gels are very difficult to destain if the gel is overstained.
>
> At the levels of EtBr I use, I can easily see less than 5 ng of DNA, so
> this is rarely a problem.  Plus,
>
> >Moreover,  is not advisable to prestain gels used to run restriction
> >fragments and plasmids.  Some say that this may cause the plasmids to
become
> >supercoiled due to intercalation (I have to check out this) and the
> >restriction fragments may not migrate in relation to its mol. wt.  (this
too
> >is unchecked information).
>
> I must disagree.  Intercalation of the ethidium is not a problem.  For
> uncut plasmids, it's easy to identify the different forms, e.g.
supercoiled
> vs. nicked.  For linerar fragments, it matters not at all whether they
have
> ethidium intercalated or not....the size standards will be in the same
> state.  Therefore accurate sizes can be determined.  And, empirically, I
> know this is not a problems--- I've been doing it this way for about 15
> years now!
>
> It is definately a time saver to prestain the gels, and frankly, the
> negative consequences you mention simple are NOT a problem.  You ought to
> try it some time!
>
> Mike
>
>
>
> >     best of luck
> >jayakumar
> >
> >----- Original Message -----
> >From: ""Michael L. Sullivan"" <mlsulliv at facstaff.wisc.edu>
> >To: <methods at hgmp.mrc.ac.uk>
> >Sent: Thursday, March 15, 2001 8:09 PM
> >Subject: Re: EtBr absorbance
> >
> >
> >> 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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