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5
Casting gradient gels
5.1
Successful gradient gel casting requires planning, timing, and practice. A full cast with the Ettan DALTsix Gel
Caster requires 420–550 ml of acrylamide stock. Polymerization begins as soon as TEMED and APS are added to
the acrylamide stock. At this point there is no time to adjust the gradient maker, or the cassettes and separators
in the gel caster. To familiarize yourself with the gel caster and gradient maker before casting gels, you should
set up the unit and carry out a practice gradient cast, substituting water for the appropriate volume of light
solution, and a mixture of glycerol and water for the appropriate volume of heavy solution.
A pore gradient gel results from mixing varying proportions of two solutions of different acrylamide
concentrations and densities, a light solution and a heavy solution. The heavy gel solution contains glycerol and
a higher concentration of acrylamide. During the gradient pouring procedure, the mixing ratio of high
concentration acrylamide solution to low concentration solution gradually changes, with the heavier (high
concentration) solution sinking below the light solution. This generates a downward gradient of increasing
acrylamide (gel) percentage.
5.2
WARNING! When using hazardous chemicals, take all suitable protective measures, such as wearing
protective glasses and gloves resistant to the chemicals used. Follow local regulations and instructions for
safe operation and maintenance of the system.
WARNING! Acrylamide is a neurotoxin. Never pipette by mouth and always wear protective gloves when
working with acrylamide solutions, IPG strips, or surfaces that come into contact with acrylamide solutions.
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Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
Preparation for gradient gel casting
Gradient casting setup
Be sure the entire gel casting system is clean, dry, and free from any polymerized acrylamide. Remove the
rubber insert from the base of the V-shaped feed channel of the caster. The caster should be placed on a
level bench to ensure that the gels and gradients are even and level.
Add a magnetic stir bar of the appropriate dimensions (20-30 mm long) to the mixing chamber and place
the unit on a magnetic stirrer. If volumes will be less than half the capacity of the unit, an identical stir bar
should be placed in the reservoir chamber as well to balance the displacement and prevent backflow into
the reservoir when the chambers are first connected.
Connect tubing to the outlet connector and pump, and adjust pump speed, if used. Connect the tubing to
the gel casting unit.
Close the connecting valve between the reservoir (heavy) and mixing (light) chambers and clamp off the exit
line.
Prepare a sufficient volume of gel overlay solution (water-saturated n-butanol). You need 1.0 ml of overlay
for each 1-mm cassette and 1.5 ml for each 1.5-mm cassette.
Make up 200 ml of displacing solution.
Make up the gel acrylamide solutions from the stock mixes, but do not add the 10% ammonium persulfate
(APS) and 10% N,N,N',N',-tetramethylethylenediamine (TEMED). See "Gradient gel solutions" on page 16.
Casting gradient gels 5
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