C.B.S. Scientific DGGEK-4801 User Manual
Page 15
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www.cbsscientific.com
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
42
SECTION 7
Maximum Well/Comb Volumes
Vertical Comb Options, deeper wells, overall length = 13.1cm
Cat. #
Tooth Depth
(mm)
# of
Teeth
Thickness
of Teeth
Width of
Teeth
Recommended
Max. Sample
Vol./Well (µl)
*
VGC-0716
19.1
16
0.75
5.2
55
VGC-0720
17.7
20
0.75
4.2
42
VGC-0722
17.7
22
0.75
4.35
25
VGC-0729
17.7
29
0.75
2.9
29
VGC-0730
17.7
30
0.75
2.8
28
VGC-1016
19.1
16
1.0
5.2
74
VGC-1020
19.1
20
1.0
4.2
55
VGC-1022
17.7
22
1.0
4.35
57
VGC-1029
17.7
29
1.0
2.9
38
VGC-1030
17.7
30
1.0
2.8
37
VGC-1516
19.1
16
1.5
5.2
110
VGC-1520
17.7
20
1.5
4.2
83
VGC-1522
17.7
22
1.5
4.35
85
VGC-1529
17.7
29
1.5
2.9
58
VGC-1530
17.7
30
1.5
2.8
56
Vertical Comb Options, short wells, overall length = 13.1cm
Cat. #
Tooth depth
(mm)
# of
Teeth
Thickness
of Teeth
Width of
Teeth
Recommended
Max. Sample
Vol./Well (µl)
*
VGC-0716-177
10
16
0.75
5.2
29
VGC-0720-177
10
20
0.75
4.2
23
VGC-0722-177
10
22
0.75
4.35
25
VGC-0729-177
10
29
0.75
2.9
16
VGC-0730-177
10
30
0.75
2.8
16
VGC-1016-177
10
16
1.0
5.2
39
VGC-1020-177
10
20
1.0
4.2
32
VGC-1022-177
10
22
1.0
4.35
32
VGC-1029-177
10
29
1.0
2.9
22
VGC-1030-177
10
30
1.0
2.8
21
VGC-1516-177
10
16
1.5
5.2
58
VGC-1520-177
10
20
1.5
4.2
47
VGC-1522-177
10
22
1.5
4.35
49
VGC-1529-177
10
29
1.5
2.9
33
VGC-1530-177
10
30
1.5
2.8
32
*
Maximum loading volume is calculated by taking 75% of the total tooth volume.