Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 66)

7.2 Reagent Preparation
The concentration and type of acrylamide to use varies for the sample being analyzed on
the DCode system, therefore, a 40% stock solution containing acrylamide and bis-acrylamide
(Bis) should be made. Reagents for casting and running PTT gels are included in the DCode
electrophoresis reagent kit for PTT, catalog number 170-9174.
For different percent crosslinking, use the equation below to determine the amount of Bis
to add. The example stock solution below is for an acrylamide/bis ratio of 37.5:1.
40% Acrylamide/Bis (37.5:1)
Reagent
Amount
Acrylamide 38.93 g
Bis-acrylamide 1.07 g
dH
2
O to 100.0 ml
Filter through a 0.45 µ filter and store at 4°C.
Polyacrylamide gels are described with reference to two characteristics:
1) The total monomer concentration (%T)
2) The crosslinking monomer concentration (%C)
%T =
gm acrylamide + gm bis-acrylamide
x 100
total volume
%C =
gm bis-acrylamide
x 100
gm acrylamide + gm bis-acrylamide
1.5 M Tris-HCl, pH 8.8
Reagent
Amount
Tris base 54.51 g
dH
2
O 150.0 ml
Adjust to pH 8.8 with 6 N HCl. Add dH
2
O to 300 ml.
0.5 M Tris-HCl, pH 6.8
Reagent
Amount
Tris base 6.0 g
dH
2
O 60.0 ml
Adjust to pH 6.8 with 6 N HCl. Add dH
2
O to 100 ml and. Store at 4°C.
10% SDS
Reagent
Amount
SDS 10.0 g
dH
2
O to 100.0 ml
Mix and store at room temperature.
10x Tris/Glycine/SDS Buffer, pH 8.3
Reagent
Amount Final Concentration
Tris base 30.3 g 0.25 M
Glycine 144.1 g 1.92 M
SDS 10.0 g 1%
dH
2
O to 1,000 ml
Mix and store at room temperature.
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