Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 67)

The table below provides the percentage acrylamide/bis needed for a particular size range.
Gel Percentage Base Pair Separation
7.5% 35–95 kD
10% 15–70 kD
15% 10–40 kD
Separating Gel–0.375 M Tris, pH 8.8
Reagent
7.5% 12% X%
40% Acrylamide/Bis 7.5 ml 12.0 ml (X%) = (A) * ml
1.5M Tris-HCl, pH 8.8 10.0 ml 10.0 ml 10.0 ml
10% SDS 0.4 ml 0.4 ml 0.4 ml
dH
2
O 17.2 ml 17.2 ml 29.2–(A) *
10% Ammonium persulfate 400.0 µl 400.0 µl 400.0 µl
TEMED 40.0 µl 40.0 µl 40.0 µl
Total volume 40.0 ml 40.0 ml 40.0 ml
Degas for 15 minutes before adding TEMED and ammonium persulfate. Cast the gel immediately
after adding the TEMED and ammonium persulfate.
* The letter A designates the volume of 40% acrylamide/bis solution required to produce the specified percent of gel (X%).
4% Stacking Gel–0.125 M Tris, pH 6.8
Reagent
Amount
40% Acrylamide/Bis 1.0 ml
0.5 M Tris-HCl, pH 6.8 2.5 ml
10% SDS 0.1 ml
dH
2
O 6.4 ml
10% Ammonium Persulfate 50.0 µl
TEMED 10.0 µl
Total volume 10.0 ml
Degas for 15 minutes before adding TEMED and ammonium persulfate. Cast the gel immediately
after adding the TEMED and ammonium persulfate.
Laemmli Sample Buffer
Reagent
Amount Final Concentration
0.5 M Tris-HCl, pH 6.8 3.1 ml 62.5 mM
100% Glycerol 6.25 ml 25%
10% SDS 5.0 ml 2%
0.5% Bromophenol blue 0.5 ml 0.01%
dH
2
O 10.15 ml
Total volume 25.0 ml
Mix and store at 4°C. Before use add 10 µl ß-mercaptoethanol to 590 µl Laemmli buffer.
Dilute sample 1:2 with Laemmli buffer.
Coomassie Blue Stain
Reagent
Amount Final Concentration
Coomassie Blue R-250 1.0 g 0.1%
Methanol 400 ml 40%
Acetic acid, glacial 100 ml 10%
dH
2
O 500 ml
Total volume 1,000 ml
Mix and store at room temperature.
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