Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 34)

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.
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 about 10–15 minutes. 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 about 10–15 minutes. 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.
To cast constant denaturing gradient gels, use the formula below to determine the volume of
0% and 100% denaturing solutions needed to achieve the desired denaturant concentration.
1. (% desired denaturant) (total gel volume needed) = ml of 100% denaturant solution
2. (total gel volume needed) - (ml of 100% denaturant) = ml of 0% denaturant solution
Example: To cast a 52% constant denaturing gel, use 30 ml total volume for a 16 x 16 cm
gel with a 1.0 mm spacer.
1. (0.52)(30 ml) = 15.6 ml of 100% denaturing solution needed
2. (30 ml)–(15.6 ml) = 14.4 ml of 0% denaturing solution needed
The table below provides the percentage acrylamide/bis needed for a particular size range.
Gel PercentageBase Pair Separation
6% 300–1,000 bp
8% 200–400 bp
10% 100–300 bp
10% Ammonium Persulfate
Reagent
Amount
Ammonium persulfate 0.1 g
dH
2
O 1.0 ml
Store at –20°C for about a week.
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