Xylanase Protocols

Introduction

This protocol is a method to follow for Xylan degredation on agarose plates.

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
• Acetone

Procedure

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 with a 2:1 mixture of Ethanol 0.05 M - Sodium Acetate in water until sample is colorless.
8. Following this protocol results in collection of ~1.0g RBB-Xylan with a collection of dye obtained ranging from 2.6-25%.
9. Added to the plates is 1% RBB-Xylan weight per volume.