Owner's manual

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.