Xylanase Protocols: Difference between revisions
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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 | ||
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#Wash the precipitate with 50ml Acetone. | #Wash the precipitate with 50ml Acetone. | ||
#Dry at room temp. | #Dry at room temp. | ||
#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 14:08, 5 May 2011
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
- 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 with 500ml 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.
- Added to the plates is 1% RBB-Xylan weight per volume.