Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 59)

Using Acrylamide and Bis-acrylamide Stock Solutions
Use these calculations to determine the necessary volumes of stock acrylamide and
bis-acrylamide solutions to produce gels of any percent and volume.
To determine the monomer and crosslinker ratios:
1. (A) is the part of total monomer that is acrylamide
A =
gm acrylamide
gm acrylamide + gm bis-acrylamide
2. (B) is the part of total monomer that is bis-acrylamide
B =
gm bis-acrylamide
gm acrylamide + gm bis-acrylamide
To determine the volume of 40% acrylamide stock solution to use:
3. Use the calculation for (A) determined above.
(A) (gel %) (final volume)
= ml of 40% acrylamide (C)
(40%) acrylamide solution
4. Use the calculation for (B) determined above.
(B) (gel %)(final volume)
= ml of 2% bis-acrylamide (D)
(2%) bis-acrylamide solution
Acrylamide/Bis Solutions (1x TBE)
Reagent
8% Gel (37.5:1) X% Gel
40% Acrylamide 7.78 ml (C) from above
2% Bis 4.27 ml (D) from above
10x TBE (see note) 4 ml 4 ml
100% Glycerol (optional) see note see note
TEMED 40 µl 40 µl
10% Ammonium persulfate 400 µl 400 µl
dH
2
O to 40 ml to 40 ml
Cast the gel immediately after adding the TEMED and ammonium persulfate.
Optional: 0.5x TBE, add 2 ml
5% glycerol, add 2 ml
10% glycerol, add 4 ml
10% Ammonium Persulfate
Reagent
Amount
Ammonium persulfate 0.1 g
dH
2
O 1.0 ml
Store at –20°C for about a week.
2x Gel Loading Dye
Reagent
Amount Final Concentration
Bromophenol blue 0.05 g 0.05%
Xylene cyanol 0.05 g 0.05%
Formamide 9.5 ml 95%
0.5 M EDTA, pH 8 0.4 ml 20 mM
Total volume 10.0 ml
Store at room temperature.
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