Xylanase Protocols

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(Procedure)
(Plate Production)
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*20ml Sodium Acetate Solution (0.675g in 20ml)
*20ml Sodium Acetate Solution (0.675g in 20ml)
*200ml 96% Ethanol
*200ml 96% Ethanol
 +
*500ml Wash solution (330ml Ethanol, 270ml Water, 1.1g Sodium Acetate)
*Acetone
*Acetone
Line 27: Line 28:
#Wash the precipitate with 50ml Acetone.
#Wash the precipitate with 50ml Acetone.
#Dry at room temp.
#Dry at room temp.
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2:1 mixture of Ethanol 0.05 M - Sodium Acetate in water until sample is colorless.
+
 
-
#Following this protocol results in collection of ~1.0g RBB-Xylan with a collection of dye obtained ranging from 2.6-25%.
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#Added to the plates is 1% RBB-Xylan weight per volume.
#Added to the plates is 1% RBB-Xylan weight per volume.

Revision as of 17:08, 5 May 2011

Contents

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
  • 500ml Wash solution (330ml Ethanol, 270ml Water, 1.1g 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 with 500ml 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.
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