Bio-Rad PROTEAN II XL Cell - Manual (Page 17)

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4.2 Casting Continuous Gels
Continuous gels are ones in which the entire gel is of one composition. This type of gel is often used in non-
denaturing (native) buffer systems.
1. Prepare the monomer solution. Combine all reagents except APS and TEMED. Degas under vacuum for
at least 15 minutes.
2. Place a comb in the glass sandwich so that the teeth are tilted at approximately a 10° angle.
3. Add APS and TEMED to the degassed monomer solution, and use a pipet and bulb to pour the solution
down the spacer nearest to the upturned side of the comb. Pour until the bottoms of all the teeth are
covered. Then adjust the comb to its proper position. Add monomer solution to fill the sandwich
completely. No overlay solution is necessary
4. Let the gel polymerize for 45 minutes to 1 hour. The gel is now ready to load and run. Remove the
comb, and rinse thoroughly with distilled water.
4.3 Casting Gradient Gels
Polyacrylamide concentration gradients, made with an external gradient former (Model 385, catalog
#165-2000), can be introduced into the PROTEAN
® II xi gel sandwich assembly either from the bottom or
from the top. A peristaltic pump is required for introduction from the bottom. Introduction from the top can
be done by gravity flow or with a peristaltic pump. Follow the gradient former instructions for formulating the
gradient. If gradients are pumped into the gel sandwich from the bottom, the low density solution (low
percent monomer) must enter first. If the gradient enters from the top, the high density solution (high percent
monomer) enters first.
Pouring Gradient Gels from the Bottom
1. After assembling the gel sandwich as described in Section 4, attach the gradient former tubing to a
gradient pouring needle (catalog #165-2007).
2. Cam the gel sandwich to the casting stand, turn the casting stand on its side, locate the bottom filling ports
directly under the space in the sandwich, and insert the botom-fill needle through the rubber gasket, so
that it protrudes about 2 mm into the sandwich. Make sure the needle opening faces one of the glass
plates.