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Denaturing Urea PAGE -Large Gel Preparation of Glass Plates

Summary: 32
Denaturing Urea PAGE - Large Gel
Preparation of Glass Plates
1. Clean glass plates and comb with soap and water. Rinse and wipe to dry with EtOH and Kimwipe.
2. Siliconize plates: Using a Pasteur pipette, add 5 drops of dichlorodimethylsilane (Aldrich D6,082-6)
to 5 mls of acetone in diposable glass tube. Mix. Pour ~2.5 mls on inside surface of long glass
plate. Spread and polish with Kimwipes. Repeat for short glass plate. Wear gloves.
3. Place long glass plate on 2 foam rectangles. Set up spacers and short glass plate. Clamp.
Pouring Gel
1. 0.4 mm (6%) 0.4-0.8 mm (6%)
Ultrapure Urea 33.6 g 48.0 g
ddH2O 27 mls 38 mls
40% Acrylamide 10.5 mls 15 mls
10X TBE 7 mls 10 mls
Total Volume (for IBI sequencer) 70 mls 100 mls
Heat in microwave (high) 15 s 20 s
Stir until dissolved.
TEMED 50 Ál 75 Ál
25% APS (0.25 g + 0.85 ml) 50 Ál 75 Ál
2. Seal glass plates: Add 1 Ál TEMED and 2 Ál 25% APS to 1 ml of gel solution. Mix. Pour down


Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida


Collections: Biology and Medicine