Xylanase Protocols

Introduction

These protocols are used to screen for Xylan degradation on agarose plates, and measure xylanase activity.

Plate Production

Materials

• 2.5g Xylan
• 2.5g RBB (Remazol Brilliant Blue)
• 60ml Water
• 20ml Sodium Hydroxide Solution (1.5g in 20ml)
• 20ml Sodium Acetate Solution (0.675g in 20ml)
• 200ml 96% Ethanol
• 1L Wash solution (660ml Ethanol, 330ml Water, 1.35g Sodium Acetate)
• Acetone

Preparing the Dyed Xylan

1. Add 2.5g RBB dye and 2.5g Xylan to 60 ml Water.
2. Add 20ml Sodium Acetate solution to RBB-Xylan solution.
   1. Add dropwise over 5 minutes while stirring at room temp.
3. After mixing add 20ml Sodium Hydroxide solution.
4. Stir for 90 minutes at room temperature.
5. Add two volumes of 96% Ethanol to precipitate the xylan-RBB.
6. Filter using a vacuum flask and watman filter paper.
7. Wash the precipitate sequentially with 1L of wash solution.
   1. The filtrate should now be colorless.
8. Wash the precipitate with 100ml 75% Ethanol.
9. Wash the precipitate with 50ml Acetone.
10. Dry at room temp.

1. Added to the plates is 1% RBB-Xylan weight per volume.