Bio-Rad Model 225 Tube Gel Casting Stand - Manual (Page 14)

Section 7
The Second-Dimension
While the first-dimension tube gels are running, cast the second dimension
slab gels according to the directions in the Mini-PROTEAN cell instruction manual.
For reproducibility of slab gel composition of multiple gels, use the Mini-PROTEAN
multi-gel casting chamber.
7.1 Preparation of the Stacking Gel
1. To prepare the stacking gel with a standard well for the second-dimension run, use
the 2-D comb pushed halfway in. Cast a narrow stacking gel (maximum 5 mm),
and fill the excess space on top of the tube gel for the equilibration buffer, if
necessary. Follow the instructions in the Mini-PROTEAN cell instruction manual.
Alternatively, when using molecular weight standards in agarose, cast the stacking
gel and overlay with water over the entire surface of the gel without using a comb.
Cast the molecular weight standards into an agarose tube gel as described in the
solutions section, and load the agarose gel piece directly onto the stacker with a
spatula after application of the first-dimension tube gel.
7.2 Removing Gel from the Tube and Loading onto the Slab Gel
1. After the first-dimension run is complete, turn off the power, disconnect the lid
and remove the tube gel module. Separate the tubes from the connectors and
sample reservoirs.
2. Attach a 1.0 ml syringe to the white end of the tube gel ejector. Draw up
Laemmli electrophoresis buffer through the ejector into the syringe.
(Preparation of the SDS-PAGE (Laemmli) running buffer is described in the
Mini-PROTEAN cell manual.)
3. Loosen the colored end of the gel ejector by unscrewing it almost all the way.
Separate the capillary tube from the sample reservoir. Insert the capillary tube
into the colored end of the unit when it is unscrewed. The tube will encounter
minor resistance and must be inserted all the way in to get a tight seal. When
the tube is all the way in, tighten the colored part to secure the tube.
4. Put equilibration or reducing buffer between the slab gel plates so that the tube
gel will slide easily between the glass plates.
5. Extrude the tube gel onto a piece of Parafilm laboratory film. When extruding
the gel, pay attention to the amount of pressure applied to the plunger. Push
hard on the plunger until the gel begins to move down the tube. Once the gel
starts moving, the resistance decreases rapidly. When you notice that the gel
has started to move, release the pressure and then push more slowly.
6. Wet the tube gel slightly with SDS sample equilibration buffer. With a clean
spatula, straighten out the gel and position it lengthwise on the Parafilm laboratory
film. Carefully slide the gel from the Parafilm laboratory film between the glass
plates and onto the slab gel.
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