Manual

28
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