Bio-Rad Model 225 Tube Gel Casting Stand - Manual (Page 7)

Section 3
Solutions
3.1 Solutions for First-Dimension
1. First-dimension acrylamide stock solution
Acrylamide/bis (30% T/5.4% C)
Acrylamide
28.38 g
Bis-acrylamide 1.62 g
100 ml
Fill to a total volume of 100 ml with distilled water. Filter and store at 4 °C in the
dark (30 days maximum).
2. 10% Triton X-100 stock (w/v) solution: Dilute 10 g Triton X-100 detergent to
a total volume of 100 ml with distilled water. Deionize overnight with 5 gm of
AG 501-X8 ion exchange resin. Conductivity should be
≤ 1 µ mho.
3. First-dimension sample buffer
8.0 M urea 5.7 g
2.0% Triton X-100 2.0 ml 10% Triton X-100 stock
5% β-mercaptoethanol 0.5 ml
1.6% Bio-Lyte 5/7 ampholyte 400 µl
0.4% Bio-Lyte 3/10 ampholyte 100 µl
Dilute to 10 ml with distilled water (warm slightly in a water bath to dissolve
urea). Do not exceed 30°C or the urea will break down. Aliquot into 0.5 ml
volumes in Eppendorf tubes. Store at -70°C.
4. First dimension sample overlay buffer
4.0 M urea 5.41 g
0.8% Bio-Lyte 5/7 ampholyte 200 µl
0.2% Bio-Lyte 3/10 ampholyte 50 µl
Bromophenol blue 500 µl of a 0.05% (w/v) Bromophenol blue
stock solution
Dilute to 10 ml with distilled water. Warm in a water bath at no hotter than 30°C
to dissolve the urea. Aliquot into 0.5 ml volumes in Eppendorf tubes. Store at
-70°C.
5. Upper Chamber Buffer (100 mM NaOH)
Dissolve 1.0 g NaOH in 250 ml distilled water and degas thoroughly for 30 minutes.
Degassing helps prevent bubbles from forming in the sample reservoirs, or in the
tubes between the gel and the sample reservoir. If bubbles form, electrophoresis is
inhibited.
6. Lower Chamber Buffer (10 mM H
3
PO
4
)
Dilute 1.36 ml concentrated H
3
PO
4
in 2 L distilled water and degas thoroughly
for 30 minutes. Degassing helps prevent bubbles from lodging on the bottom of
the tubes.
4