Xylanase Protocols

Introduction
Use this protocol to screen for Xylan degradation on agarose plates.

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


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