Casting Homogeneous Gels - GE HEALTHCARE Ettan DALTsix User Manual

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Casting homogeneous gels

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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10 Allow the homogeneous gels to polymerize for at least one hour before disassembling the caster. If the
Ettan DALTsix Electrophoresis System User Manual 80-6492-49 Edition AC
Be sure the entire gel casting system is clean, dry, and free from any polymerized acrylamide.
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 overlay for each 1.5-mm cassette. Mixing 1-mm and 1.5-mm
cassettes, in one casting is not advised.
If casting using the displacement method, make up 200 ml of displacing solution (0.375 M Tris-Cl pH 8.8, 30%
(v/v) glycerol, bromophenol blue).
For a full 6-gel, 1.5-mm cassette set, make up 600 ml of acrylamide gel stock solution without ammonium
persulfate (APS) or N,N,N',N'-tetramethylethylenediamine (TEMED). For a full 6-gel, 1-mm cassette set, make
up 450 ml of acrylamide gel stock solution as above. This amount of gel solution will provide you with
sufficient volume to cast gels using either the filling channel or a peristaltic pump.
Assemble the gel caster as described in the proceeding section; the caster should be placed on a level bench
or on a leveling table so that gel tops are level.
Add the appropriate volumes of APS and TEMED only when ready to pour the gels, not before. Once these
two components are added, polymerization begins and the gel solution should be completely poured within
10 min.
Pour the gel solution into the filling channel. If displacement casting is being used, the flow rate on the
peristaltic pump should be increased slowly to the desired speed to avoid introducing any air bubbles.
For displacement casting, pump the gel solution into the caster until it is about 7–11 cm below the final
desired gel height. Stop the flow of acrylamide and transfer the feed tube to the container holding the
displacement solution. Restart the pump to displace the gel solution in the tubing and the V-trough at the
bottom of the caster. The remaining acrylamide solution is forced into the cassettes to the final gel height.
When the final desired gel height is reached, stop the pump and clamp off the tubing near the barbed fitting
at the bottom of the caster.
For filling channel casting, pour the gel solution into the channel until the solution reaches the final desired
height, about 1 cm below the top edge of the short plate.
The amount of gel solution required in either case will be ~550 ml for six 1.5-mm gels and ~425 ml for six
1-mm gels.
Immediately pipette the water-saturated n-butanol onto each gel. Avoid wetting adjacent plastic surfaces
of the caster with n-butanol.
peristaltic pump outlet tubing was connected directly to the caster, it should not be removed until
polymerization is complete. Do not leave the water-saturated n-butanol on the gels overnight as long term
exposure of acrylic to butanol will damage the caster.
Casting homogeneous gels 4
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