Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 51)

CSGE Analysis
For a different percent crosslinking, use the equation in the heteroduplex analysis reagent
preparation to determine the amount of crosslinker to add. The example stock solution below
is for an acrylamide/PDA ratio of 99:1.
40% Acrylamide/PDA (99:1)
Reagent
Amount
Acrylamide 198.0 g
PDA or BAP 2.0 g
dH
2
O to 500.0 ml
Filter through a 0.45 µ filter. Store at 4°C.
10x TTE Buffer
Reagent
Amount Final Concentration
Tris base 107.8 g 0.89 M
Taurine 18.8 g 0.15 M
EDTA 1.9 g 5 mM
dH
2
O to 1,000 ml
Mix. Autoclave for 20–30 minutes. Store at room temperature.
10% Acrylamide/PDA Gel
Reagent
Amount Final Concentration
40% Acrylamide/PDA (see note) 10.0 ml 10%
10x TTE 2.0 ml 0.5x
Formamide 6.0 ml 15%
Ethylene Glycol 4.0 ml 10%
dH
2
O 17.6 ml
TEMED 40.0 µl
10% Ammonium persulfate 400.0 µl
Total volume 40.0 ml
Mix. Cast the gel immediately after adding the TEMED and ammonium persulfate.
Note:
For 6% acrylamide gel use 6 ml of a 40% stock.
For 8% acrylamide gel use 8 ml of a 40% stock.
For 12% acrylamide gel use 12 ml of a 40% stock.
1x TTE Lower Chamber Running Buffer
Reagent
2 L Buffer 7 L Buffer
10x TTE buffer 200 ml 700 ml
dH
2
O 1,800 ml 6,300 ml
0.25x TTE Upper Chamber Running Buffer
Reagent
400 ml Buffer
10x TTE buffer 10 ml
dH
2
O 390 ml
5.3 Gel Volumes
The table below provides the required volume for the gel size and spacer thickness.
Spacer Thickness 20 x 20 cm Gel
0.75 mm 30 ml
1.00 mm 40 ml
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