Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 58)

6.2 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 an SSCP gel are included in the
DCode electrophoresis reagent kit for SSCP, catalog number 170-9172.
For a 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)
%T =
g acrylamide + g bis-acrylamide
x 100
Total Volume
2) The crosslinking monomer concentration (%C)
%C =
g bis-acrylamide
x 100
g acrylamide + g bis-acrylamide
10x TBE Buffer
Reagent
Amount Final Concentration
Tris base 108 g 0.89 M
Boric acid 55 g 0.89 M
0.5 M EDTA, pH 8.0 40 ml 20 mM
dH
2
O to 1L
Mix. Autoclave for 20–30 minutes. Store at room temperature.
40% Acrylamide/Bis Solutions (1x TBE)
Reagent
6% Gel 8% Gel 10% Gel 12% Gel
40% Acrylamide/Bis 6 ml 8 ml 10 ml 12 ml
10x TBE (see note) 4 ml 4 ml 4 ml 4 ml
100% Glycerol (optional) see note see note see note see note
TEMED 40 µl 40 µl 40 µl 40 µl
10% Ammonium persulfate 400 µl 400 µl 400 µl 400 µl
dH
2
O to 40 ml to 40 ml to 40 ml to 40 ml
Add dH
2
O to 40 ml and mix. Cast the gel immediately after adding the TEMED and ammonium
persulfate.
Note: 0.5x TBE, add 2 ml.
5% glycerol, add 2 ml.
10% glycerol, add 4 ml.
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