C.B.S. Scientific DGGEK-2401 User Manual
Page 28
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Cipher DGGE Instructions 2/28/14
SECTION 6
Recommended Buffers and Reagents
Warning: Do not mix buffer in the DGGE tank!
6 1 Stock Solutions
Acrylamide Stock Solution 40% Acrylamide/Bis (37 5:1)
For 100ml use the following:
38.93 g of acrylamide
1.07 g of Bis-acrylamide
Add dH
2
O (or RO-H
2
O) to 100ml. Do not autoclave
50x TAE Gel Running Buffer
For 1 liter use the following:
242g Tris Base
57.1ml Glacial acetic acid
100ml .5 M EDTA, pH 8.0
Add dH
2
O (or RO-H
2
O) to 1 liter
Denaturant Stock Solution (7 5% gel)
For 100ml use:
Add dH
2
0 (or RO-H
2
O) to 100ml
Store denaturant stock solutions at 4º C in amber bottles
Polymerization Catalysts:
1. TEMED (10ul/ml gel solution)
2. APS Stock Solution (.6ul/ml gel solution)
For 50 ml of APS stock (10%) use 5 g of ammonium persulphate and water to
50ml. Store at 4ºC.
Neutral Loading Solution
For preparation of 10ml:
20% Ficoll or sucrose, 2 g of Ficoll 400 or sucrose
10mM Tris-HCl, pH 7.8, 100ul of 1.0 M Tris-HCl, pH 7.8
1mM EDTA, 20 ul of 0.5 M EDTA, pH 8.0
0.1% dye (BPB), 10 mg of dye
Water to 10ml
Percentage
10
20
30
40
50
60
70
80
90
100
40% Acrylamide/
Bis (ml)
18 8 18 8 18 8 18 8 18 8 18 8 18 8 18 8 18 8 18 8
50% TAE Buffer
(ml)
2
2
2
2
2
2
2
2
2
2
Formamide (ml)
4
8
12
16
20
24
28
32
36
40
Urea (g)
4 2
8 4
12 6 16 8
21
25 2 29 4 33 6 37 8
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