Methods and reagents: Pouring sequencing gels the old fashioned way

Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet.  This month's column describes some different ways for pouring DNA
sequencing gels, and there are some other tips as well. For details on how to
partake in the newsgroup, see the accompanying box.

The conventional method for pouring a polyacrylamide sequencing gel involves
placing mylar spacers between two glass plates of approximately 30 x 40 cm and
clamping along the long sides to make a sandwich, then taping the plates
together on both sides and along the bottom.  The sandwich is filled with
polyacrylamide solution by pipeting it into the open end while tipping the
assembled plates at about 45 degrees towards you. As the assembly fills,  the
glass plates are slowly moved into a horizontal position, a top spacer fitted
into the top and clamps placed over it.

Stopping the leaks
******************
Sticky tape.
Recently, Rob Jordan (rjordan@u.washington.edu) wrote that he was having some
difficulties in getting replacement tape used for assembling the sequencing gel
sandwich the old fashioned way, and asked for some advice as to which is the
best tape.  Some people suggested special sequencing plate sealer tape sold by
various companies including Life Technologies. However, rolls of tape sold by
companies specifically for sequencing gels are usually very expensive.  Netters
have found that many other kinds of tape can be used, including 3M Scotch [TM]
brand polyester electrical tape #56 or polytetrafluoroethylene (PTFE) film tape
(extruded) #5490, standard plastic boxing tape, polyvinyl chloride (PVC) tape,
teflon tape, masking tape or the like, which can be found at your local office
supply store and cost much less.

However, netters also warn that some particular types of tape are really not
suitable because they do not make leak-proof seals and the unpolymerized
acrylamide solution is more likely to leak out from the edges, even if they
have been well sealed with a double layer of tape. Therefore, the selection of
tape is a matter of trial and error.

Popular alternatives.
Most people have now switched away from using tape altogether and suggest that
the many hours spent taping glass plates is a complete waste of time. Instead
these people use two mylar side spacers and one bottom spacer with plenty of
bulldog clips around the three sides of the sandwich without any taping.  One
person suggested using a 1 cm wide Whatman 3MM [TM] filter paper as a bottom
spacer because it eliminates many leaks from occurring through the bottom of
the sandwich and it need not be removed before the run.  Electrical current can
pass through the buffer soaked paper and the Whatman insert should not alter
the electrophoresis run [1,2]. Others say that this is okay for manual
sequencing, but could cause problems for automated sequencers.

Another netter suggested making plugs of a higher percentage polyacrylamide on
the sides before pouring the gel. This can be done by using paper spacers on
all three sides and leaving a short wick of Whatman paper protruding from the
end of the plates. The idea is to allow liquid acrylamide to wet the entire
insert by dripping it onto the wick. However, this adds several steps to the
process and is very time consuming, because extra time is needed for the plugs
to polymerize. Some say that this still requires at least some taping of the
plates.

Still another suggestion was to seal only the bottom of the gel or perhaps only
the corners with agarose or a polyacrylamide solution containing a little more
ammonium persulfate and TEMED.  In addition, someone proposed that the bottom
of the sandwich can be covered with plasticine modeling clay [3], or that the
plates can be held together with a pre-fabricated `wrap-around' device or cuff
purchased from a scientific supply company. The problem with these, however, is
that the cuffs are usually not universal, being designed to fit a particular
gel system, and will therefore not fit others.

Glue.
An alternative to tape and clamps is to seal the sandwich on three sides with
glue from a hot glue gun, which can be bought from a hobby or craft store.
After placing a bead of hot glue around the plates, the glue cools, setting up
a leak barrier within the cracks of the plates in just a few minutes. [4] One
person wrote that depending upon how liberally one applies the glue, it takes
about 4-10 inches of a hot glue stick to seal a set of sequencing plates.  This
costs about 8-10 cents per gel, which is somewhat less than tape, but it takes
a little longer to apply the glue than tape.  It was noted that the most
important tip for applying hot glue is to be sure the correct angle of the glue
gun tip to the edge of the plate is made such that the glue flattens out as it
is extruded from the tip, and that the glue should be applied as one continuous
bead.  After the gel has polymerized, the glue can easily be removed by picking
the top end free with a razorblade or finger nail and peeling the whole string
off in one hand motion.  Netters say that different types of glue sticks should
be tested first because some are not as sticky as others. An added advantage is
that the glue will not stick to latex gloves as would tape.

The outcome.
After trying many different brands of tape without success, and attempting some
other methods, Rob Jordan settled on the Otter [TM] gel casting capillary
apparatus from Owl Scientific, which allows the liquid to fill the sandwich by
capillary action while sliding the glass plates together horizontally [5-8].
However, he wrote that following the instruction manual supplied is not the
best method.  A better method is to place only enough gel solution on the
plates as is necessary.  Using an automatic pipetter fitted with a 25 ml pipet,
a bead of about 5 ml is allowed to run along the advancing edge of the top
plate.  The top plate is then advanced only a few cm while the liquid fills the
space between the plates and until the edge of the solution starts to pull away
from the top or bottom edges. When that happens, the whole process is repeated
until the sandwich is completely full.  He also advises that a few practice
tries with water are useful.

Bubble trouble
**************
One major problem with pouring polyacrylamide gels is the formation of small
bubbles.  Some netters suggest that if a bubble cannot be removed by retreating
the glass plate slightly, it is sometimes easier to fish it out after the
plates are filled, but before the gel has solidified. As tilting the plates
rarely works, and tapping the glass with a metal object can introduce
microscopic nicks into the glass, which later act a nucleation sites for
bubbles the next time you pour a gel (not to mention the annoying sound it
makes), this can be done more easily with a bubble hook composed of a long
piece of spacer material with a crook shape at one end.  Bubble hooks are
available from some companies, but netters suggest making your own out of some
leftover spacer material or from a piece of X-ray film.  If an old piece of
photographic film is used, the gelatin emulsion should be stripped off with 0.1
M NaOH first. To remove a bubble, simply slide the film into the liquid until
the bubble is hooked, then slide it out again.

Cheaper PAGE runs
*****************
Netters are always looking for ways to cut the cost of supplies.  As most
commercial polyacrylamide gel electrophoresis (PAGE) units have a lower buffer
chamber that holds several times more volume than the upper buffer chamber,
some companies suggest that researchers re-use the lower buffer several times,
but that the upper buffer chamber must be filled with freshly prepared buffer
each time you run the gel.  However, netters strongly suggest that this not be
done because the pH of the buffer drops dramatically after only one or two runs
and this has quite an effect on protein migration in the bottom of the gel.
This was demonstrated by one person who tested the lower buffer with
bromophenol blue indicator.  After only two to three re-uses, the buffer turned
yellow indicating that the pH had dropped below 3.0.  As a major cost of the
buffer is the glycine or tricine added to the Tris-containing electrophoresis
buffer, one idea proposed to cut cost is to use a different buffer in the upper
and lower chambers.  By filling the lower buffer chamber with the Tris buffer
solution less the glycine and SDS, many more gels can be run for less cost. In
addition, it is much easier to make up a large batch of Tris buffer, which can
be stored at room temperature for months, rather than risking storage of large
quantities of glycine buffer. Another advantage is that there is little or no
foaming to deal with.

An interesting side-effect, however, is that when longer gels are partly
destained, there is a large frown evident on the bottom of the gel which could
be from leaching of the SDS from the bottom of the acrylamide into the buffer.
Netters say that this has no consequence on protein resolution after complete
destaining, however, and can generally be ignored.

References
**********

[1] Wahls, W. P. and Kigzette, M. (1988)
    BioTechniques 6, 308-309

[2] Law, J. C. and Ferrell, R. E. (1994)
    BioTechniques 17, 850

[3] Bollet, C., Treyssac, F. and De Micco, P. (1993)
    BioTechniques 15, 387

[4] Kyllo, J. H. et al. (1994)
    BioTechniques 16, 792

[5] Garoff, H. and Ansorge, W. (1981)
    Anal. Biochem. 115, 450-457

[6] Darnay, B. G. and Engel, M. (1992)
    BioTechniques 13, 859

[7] Chang, Y. and Wu, G. E. (1992)
    BioTechniques 12, 78

[8] Lashkari, D. A., Norgren, R. M. and Davis, R. W. (1995)
    BioTechniques 18, 625-626

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Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are
those of the author and do not reflect the opinion of the National Cancer
Institute or the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
redistribution, sale, or otherwise without written permission from the author.

You found this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jul96.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1996. Methods and reagents - Pouring sequencing gels the old
fashioned way. Trends in Biochemical Sciences 21(7):273-274.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh@ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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