PreparationofPolyacrylamideGels

1. Prepare 20X TBE as:

216 g Tris Base
110 g Boric Acid
80 mL 500 mM EDTA, pH 8.0
700 mL ddH2O
Mix. Bring volume to 1 L. Autoclave.

2. Prepare Acrylamide solution as:

6 % Acrylamide 60.0 g Acrylamide
0.25 % Bis-Acrylamide 2.5 g Bis-Acrylamide
8 M Urea 422.0 g Urea
0.5 X TBE 25 mL 20X TBE
500 mL ddH20
Mix. Bring volume to 1 L. Filter. Store at 4 C in the dark.

3. Wash glass plates on both sides with soap. Rinse well with water. Rinse well with distilled water. Hold vertical and douse each side with 95% Ethanol to promote drying. Stand on edge and dry with Kim-Wipe.

4. Lay the plates flat. Etch one side of each plate to mark it as 'Siliconized.' Spread 100 mL Sigmacote solution on each plate and distribute with a small Kim-Wipe. 

5. Allow to dry for 2 min. Place spacers on long plate. Place short plate on top providing a 2 mm gap is provided at the bottom. Clamp the bottom 2/3 with 3 bulldog clamps.

6. Prop up the top edge of the plates with a 25 mL pipet to provide a shallow slope from top to bottom.

7. Mix the appropriate amount of acrylamide solution in a 60 mL syringe. As a guide:

100 mL Acrylamide Solution
600 ml Fresh (2 weeks) 10% Ammonium Persulfate
30 ml Fresh Temed

8. Mix by inversion. Quickly apply 3 mL across the top of the plates and allow to run between the plates.

9. Continue to provide acrylamide to the top of the plates as it runs in. DO NOT allow the top to run dry or bubbles will form. DO NOT overfill. Tap any slow-running spots to quicken flow and avoid bubble formation.

10. When acrylamide solution fills the space between the plates, remove the 25 mL pipet and lay the plates flat. Insert comb teeth up and clamp. Layer excess acrylamide over the comb and across the bottom lip. Polymerize for 30 min.