Bio-Rad PROTEAN II XL Cell - Manual (Page 15)

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Section 4
Casting the Gels
4.1 Casting Discontinuous (Laemmli) Gels
Discontinuous gels consist of a resolving or separating (lower) gel and a stacking (upper) gel. The stacking
gel acts to concentrate large sample volumes, resulting in better band resolution than is possible using the
same volumes on a gel without a stack. Molecules are then completely separated in the resolving gel. The
most popular discontinuous buffer system is that of Laemmli. This formulation is included in the Appendix.
1. Prepare the monomer solution by combining all reagents except ammonium persulfate (APS) and
TEMED (see Section 14.1 for formulations). Deaerate the solution under vacuum for at least 15 minutes.
2. Place a comb completely into the assembled gel sandwich. With a marker pen, place a mark on the
glass plate 1-2 cm below the teeth of the comb. This will be the level to which the separating gel is
poured. Remove the comb.
3. Add APS and TEMED to the deaerated monomer solution, and pour the solution to the mark, using a
glass pipet and bulb. The easiest way to pour is to flow the solution down the middle of the outside
plate of the gel sandwich. Another way to pour is to flow the solution down the side of one of the
spacers. An alternative method is to use a syringe and Tygon tubing to load the solution from near the
bottom of the sandwich. In all cases, pour the solution smoothly to prevent it from mixing with air.
4. Immediately overlay the monomer solution with water, water-saturated isobutanol, or t-amyl alcohol. The
advantage of using isobutanol or t-amyl alcohol is that the overlay solution can be applied rapidly with a
Pasteur pipet and bulb because very little mixing will occur. If water is used to overlay, it must be done
using a needle and syringe, using a steady, even rate of delivery to prevent mixing.
5. Allow the gel to polymerize for 45 minutes to 1 hour. Rinse off the overlay solution completely with
distilled water. This is especially important with alcohol overlays. Do not allow alcohols to remain on the
gels more than 1 hour, or dehydration of the top of the gel will occur.
Note: It is sometimes convenient to cast the separating portion of a discontinuous gel the afternoon
before casting the stacking gel and running the gel. If the stacking gel is to be cast the following day,
place approximately 5 ml of 1:4 diluted stock solution B (see Section 14.1) on top of each separating gel
after rinsing with deionized water. This will prevent dehydration of the separating gel during overnight
storage a room temperature.