These protocols are used to screen for Xylan degradation on agarose plates, and measure xylanase activity.
- 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)
Preparing the Dyed Xylan
- Add 2.5g RBB dye and 2.5g Xylan to 60 ml Water.
- Add 20ml Sodium Acetate solution to RBB-Xylan solution.
- Add dropwise over 5 minutes while stirring at room temp.
- After mixing add 20ml Sodium Hydroxide solution.
- Stir for 90 minutes at room temperature.
- Add two volumes of 96% Ethanol to precipitate the xylan-RBB.
- Filter using a vacuum flask and watman filter paper.
- Wash the precipitate sequentially with 1L of wash solution.
- The filtrate should now be colorless.
- Wash the precipitate with 100ml 75% Ethanol.
- Wash the precipitate with 50ml Acetone.
- Dry at room temp overnight.
- Add 1% RBB-Xylan (weight per volume) to your agar media of choice after autoclaving.
- Pour the plates immediately being sure to swirl frequently.
- Drop 5μL of overnight culture onto the plates
- Incubate for two days
- Observe clearing zones around xylanase producing cultures.