Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 33)

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Section 7
Preparation of Electrophoresis Buffers and Acrylamide Stock
Solutions for SDS-PAGE
The Model 491 prep cell has three buffer reservoirs: one for the lower electrophoresis buffer (~3 liters), one
for upper electrophoresis buffer (~600 milliliters) and one for the elu tion buffer (~900 milliliters). For SDS-
PAGE all three reservoirs contain the same buffer (Laemmli buffer system). It is recommended that 6.0 liters
of buffer be pre pared for each preparative run. Premixed liquid acry lamide (concentrated solution) and
premixed electrophoresis buffers (10x) are available. (See product information Section.)
7.1 Separating (Resolving) Gel Buffer Stock
1.5 M Tris-HCl pH 8.8
Dissolve 27.23 grams Tris base in approximately 100 ml deionized water.
Adjust to pH 8.8 with 6 N HCl.
Make to 150 ml with deionized water and store at 4 ˚C.
7.2 Stacking Gel Buffer Stock
0.5 M Tris-HCl, pH 6.8
Dissolve 6 grams Tris base in approximately 60 ml deionized water.
Adjust to pH 6.8 with 6 N HCl.
Make to 100 ml with deionized water and store at 4 ˚C.
7.3 Sample Buffer (SDS-Reducing Buffer)
Deionized water 3.8 ml
0.5 M Tris-HCl, pH 6.8 1.0 ml
Glycerol 0.8 ml
10% (w/v) SDS* 1.6 ml
0.5% bromophenol blue (optional) 0.4 ml
Total volume 7.6 ml
Add 50 µl beta-mercapto-ethanol to 950 µl of sample buffer prior to use. Dilute the sample at least 1:4 with
sample buffer, and heat at 95˚C for 4 min.
*To make a 10% SDS solution: Dissolve 10 g of SDS in deionized water with gentle stirring and bring to 100
ml with water.
SDS reducing buffer is 0.06 M Tris-HCl, pH 6.8, 2% SDS, 5% beta-mercapto-ethanol, 10% glycerol and
0.025% bromophenol blue.
Very dilute samples can often be adequately reduced with a two-fold dilution (1:1) with sample buffer.
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