Bio-Rad Model 225 Tube Gel Casting Stand - Manual (Page 8)

3.2 Preparation of First-Dimension Tube Gels
1. First-dimension gel monomer solution
8.0 M urea 5.5 g
4% acrylamide (total monomer) 1.33 ml acrylamide stock
2% Triton X-100 2.0 ml 10% Triton X-100
1.6% Bio-Lyte 5/7 ampholyte 0.400 ml Bio-Lyte 5/7 ampholyte
0.4% Bio-Lyte 3/10 ampholyte 0.100 ml Bio-Lyte 3/10 ampholyte
1.97 ml distilled water
0.01% ammonium persulfate 10 µl 10% ammonium persulfate (make
this solution fresh daily)
0.1% TEMED 10 µl TEMED
This makes 10 ml total volume, enough to cast one set of gels using the casting
tube. Make this solution fresh. Weigh out the urea. Add all other ingredients except
the last two, which are the polymerization catalysts. Dissolve the urea by warming
the vessel in warm water (
≤ 37°C) with swirling. Degas the solution thoroughly for
15 minutes. Add the APS and TEMED, and swirl gently and briefly. Cast the gels as
directed in Section 5.
3.3 Solutions for Second-Dimension
1. SDS sample equilibration buffer
0.0625 M Tris HC1, pH 6.8 12.5 ml 0.5 M Tris/HCl, pH 6.8
2.3% (w/v) SDS 23 ml 10% (w/v) SDS
5.0% (v/v) β-mercaptoethanol 5 ml
10% glycerol (w/v) 8 ml
Bromophenol blue 2.5 ml 0.05% (w/v) stock solution
Distilled water 49 ml
100 ml
2. 1% agarose in SDS sample buffer (optional, refer to Section 7.2)
Prepare SDS equilibration buffer without
β-mercaptoethanol. Melt 1 g
agarose/100ml.
Add
β-mercaptoethanol to 5%.
3. Solid molecular weight standard preparation
Prepare a 1:100 dilution of molecular weight standard in molten 1%
agarose in SDS equilibration buffer. Cast a standard agarose gel in a mini
2-D tube gel. Let sit at room temperature, and extrude the gel onto
Parafilm laboratory film. Cut into 0.6 cm lengths. The gel slices can be
stored in Eppendorf tubes at -20° C.
Volume of the 0.6 cm molecular weight standard = πr 2 x length
= π (0.05 cm) 2 x 0.6 cm
≈ 5 µl
For second dimension gel solutions, refer to the Mini-PROTEAN cell
instruction manual.
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