Solving the Red Rain problem on the ABI 377 48cm well to read gels

There has been a discussion on the bionet.genome.autosequencing newsgroup
regarding the observation of red streaks near the end of the 10+ hour
48 cm well to read gels.

Steve Toth made the following observations and we confirmed his 
conclusions after testing his idea.

From: IN%"toth@cric.com"  4-DEC-1996 18:17:23.45
To:	  IN%"autoseq@net.bio.net"
Subj: RED RAIN: possible simple solution

G'day All

     There has been a lot of concern over the reason, course and
solution for the infamous "RED RAIN" problem that has been appearing 
in ABI-377 gels.  Following are my observations and possible simple 
solution to eliminate the course.  For those that do experience this 
"RED RAIN"  (and due to the course not everyone is going to be as 
likely to observe the problem) please try this simple solution and let 
me know, as well as others, whether this really is a solution.

     Reason: I guess most people will have observed by know the the
reason for the "RED RAIN" is that bubbles get in front of the optics.  
You can observe these bubbles by looking at you gel once it has 
finished running (and you have "RED RAIN").  Remove the heater plate 
and lift up the cross bar in front of the optics.  These bubbles have 
migrated (floated) from the bottom of the gel up.  These bubbles are 
between the gel and the glass plate as opposed to bubbles in the gel 
itself.  You can induce getting these bubbles i.e. make "RED RAIN" 
(you can do this on a freshly used gel) by just lightly sticking a 
spatula in at the bottom of your plate, jiggle it around, creating 
bubbles in the plate then put it back in the ABI-377 and do plate 
check.

     Course:  The course for the above problem, i.e. the reason
the bubbles get there is due to drying of the gel as the gel is 
polymerising.   This courses the gel to wrinkle at the bottom.   For 
the polymerisation to give a good gel it has to have the correct media 
(water) volume and stability (no evaporation) to work.  As the gel is
polymerising and drying these wrinkles appear where bubbles can be 
introduced and then these bubbles migrate up through these wrinkles.  
I had gotten to the point where I had 100% success rate for predicting 
which gels would produce "RED RAIN" (out of 15 gels run).

     Solution:  I have found that by putting a wet paper towel on the
bottom of the gel once it is poured, either on the Otter or as I do it 
by tapping the solution down.  This will keep the humidity up for the 
gel and therefore will not have evaporation.  Observing the gel that 
is made this way you should observe that there is no wrinkling of the 
gel at the bottom, and even after the gel has been run the bottom will 
still be nice and even.

     Well I hope this may help clear up some problems for all out
there.  I am still using the wet towel method and haven't seen "RED 
RAIN."  Hopefully this was a simple solution and not just that it 
happened to be good gels (hard to have good controls to totally test 
this other than statistics and observation).  Let us all know what you 
all think.

Cheers

Stephen Toth
Genome Therapeutics Corp.
100 Beaver St.
Waltham, MA  02154
tel:  617-893-5007 X2551
      617-398-2551 (direct line)
fax:  617-893-9535
      617-642-0310 (back-up)
toth@cric.com

Steve,
        IT WORKS!!!! No more red rain!!!!  What we did was to follow your
advice and within various times after pouring the gels, covered the lower
end of the glass plates with a water moistened paper towel.  We made
sure not to have the towel touch the bottom of the glass so as to avoid
any capillary action that would cause the gel mix to be drained from
the bottom of the gel, and like magic, we no longer are having the red
rain problem.  Because it's pretty dry here in Oklahoma, we also are
covering the draped paper towel with a piece of Saran Wrap to prevent
evaporation of water off the towel.  We've waited as long as 45 minutes
prior to adding the moist towel but 45 minutes seems too long as some
slight red rain did occur.  It's just as easy to add the water moistened
paper towel within 5 minutes after pouring, as long as care is taken to avoid
drawing out any of the gel from the bottom by capillary action prior to it
being completely polymerized.   Then, after about 2 hours, we seal both
the bottom and top with a moist paper towel wrapped with Saran Wrap and
store the gels for at least 24 hours in a drawer prior to use as we have
done for years.

        It's my semi-educated guess that when the gel dries out at the
bottom (or top for that matter) and sample with "salts" and heat is applied,
the gel shrinks slightly and pulls away from the glass.  Supprisingly this
occurs several cm from the end and the void between the glass and the
polymerized but shrunken gel is filled with water/buffer that has a different
refractive index than the gel itself.  Thus the "bubbles" develop and we
see "red rain".

        As far as I'm concerned, placing the moist paper towel over the
lower end of the gel and avoiding contact with the polymerizing gel mix
at that end has SOLVED the "red rain" problem.

Thanks and a tip-of-the-hat to you for suggesting the earlier addition of
the moist paper towel to keep the area at the bottom of the gel from drying
out prior to complete polymerization of the gels.

Cheers........broe
***************************************************************************
Bruce A. Roe, Ph.D    Department of Chemistry and Biochemistry
                      University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610;  FAX: (405) 325-7762;  e-mail:  broe@ou.edu
********************** http://www.genome.ou.edu/ ************************** 

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