Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 37)

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8.4 Preparative SDS-PAGE Separating and Stacking Gels
(The following calculations are based on the preparation of a full length sepa rating gel of ~11 cm with a ~2.5
cm stacking gel in the 28 mm ID gel tube)
Table 12. Calculating %T.
Monomer
Concentration
Separating Gel
%x = %T
Stacking Gel
4% T
Acrylamide/bis (30% T/2.67%C
stock solution) c ml 1.3 ml
Deionized water d ml 6.2 ml
0.5 M Tris HCI, pH 8.8 10.0 ml –
1.5 M Tris HCI, pH 6.8 – 2.5 ml
10 % ammonium persulfate 100 µl 50.0 µl
TEMED 10 µl 10.0 µl
Total monomer 40 ml 10.0 ml
c ml: Calculate the volume of acrylamide/bis stock required for the desired total monomer
concentration with the following formula:
c ml = (x%) (40 ml)/% acrylamide stock solution
d ml: Calculate the volume of water required for the desired total monomer concentration with the
following formula:
d ml = 40 ml – 10 ml – 100 µl – 10 µl – c ml = 29.89 ml – c ml
The volume of the stacking gel should be one-and-one-half to twice the sam ple volume to be
electrophoresed. Large sample volumes will require large stacking gels, which will limit the length of the
resolving gel.
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