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Bio-Rad Mini-PROTEAN 3 Multi-Casting Chamber User Manual

Page 8

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Premixed Gel Casting Buffers

161-0798

Resolving Gel Buffer 1.5 M Tris-HCl

, pH 8.8, 1L

161-0799

Stacking Gel Buffer 0.5 M Tris-HCl

, pH 6.8, 1 L

Appendix A
Estimated Volume of Acrylamide for
12 Mini-PROTEAN 3 Gels

The first time gels of a certain thickness are cast, it is necessary to empirically determine

the required volume of acrylamide. Assemble the stack, and inject a measured volume of
water through the stopcock. Prepare this volume (+5 ml) of acrylamide.

As a guideline, this is the estimated volume of acrylamide for 12 Mini-PROTEAN 3 gels:

Spacer plates

Volume for 12 gels

Volume to prepare

0.5 mm

70 ml

75–80 ml

0.75 mm

80 ml

85–90 ml

1.0 mm

100 ml

105–110 ml

1.5 mm

140 ml

145–150 ml

Sample calculation: preparing twelve 1.0 mm 4–20% gradient gels:

Casting twelve 1.0 mm gels requires 100 ml; prepare 110 ml.

Divide the total volume by 2 to get the volume required for each chamber. (For this example,
make 55 ml for the light chamber and 55 ml for the heavy chamber.)

Solution Volume Calculations:
Light Solution (4%)
Acrylamide
30% stock solution
(30%) (X ml) = (4%) (55 ml)

X = 7.3 ml

Tris-Cl Buffer
1.5M Tris-Cl stock buffer pH 8.8
(1.5M) (X ml) = (.375M) (55 ml)

X = 13.8 ml

Water
(55 ml)–(7.3 ml + 13.8 ml) = X

X = 34 ml

APS
10% Stock solution
(500 µl) / (100 ml) = (X µl) / (55 ml)

X = 275 µl

TEMED
10% of the APS volume; (275 µl) / 10 = X

X = 27.5 µl

Heavy Solution (20%)
Acrylamide
30% Stock solution
(30%) (X ml) = (20%) (55 ml)

X = 36.7 ml

Tris-Cl Buffer
1.5M Tris-Cl Stock Buffer pH 8.8
(1.5M) (X ml) = (.375M) (55 ml)

X = 13.8 ml

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