Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 18)

Polyacrylamide gels are described by 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
50x TAE Buffer
Reagent
Amount Final Concentration
Tris base 242.0 g 2 M
Acetic acid, glacial 57.1 ml 1 M
0.5 M EDTA, pH 8.0 100.0 ml 50 mM
dH
2
O to 1,000.0 ml
Mix. Autoclave for 20–30 minutes. Store at room temperature.
The table below provides the percentage acrylamide/bis needed for a particular size range.
Gel Percentage Base Pair Separation
6% 300–1000 bp
8% 200–400 bp
10% 100–300 bp
0% Denaturing Solution
6% Gel 8% Gel 10% Gel
12% Gel
40% Acrylamide/Bis
15 ml 20 ml 25 ml 30 ml
50x TAE buffer 2 ml 2 ml 2 ml 2 ml
dH
2
O 83 ml 78 ml 73 ml 68 ml
Total volume 100 ml 100 ml 100 ml 100 ml
Degas for 10–15 minutes. Filter through a 0.45 µ filter. Store at 4°C in a brown bottle for
approximately 1 month.
100% Denaturing Solution
6% Gel 8% Gel
10% Gel 12% Gel
40% Acrylamide/Bis 15 ml 20 ml 25 ml 30 ml
50x TAE buffer 2 ml 2 ml 2 ml 2 ml
Formamide (deionized) 40 ml 40 ml 40 ml 40 ml
Urea 42 g 42 g 42 g 42 g
dH
2
O to 100 ml to 100 ml to 100 ml to 100 ml
Degas for 10–15 minutes. Filter through a 0.45 µ filter. Store at 4°C in a brown bottle for
approximately 1 month. A 100% denaturant solution requires re-dissolving after storage. Place
the bottle in a warm bath and stir for faster results.
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