Richard Lab: NDF
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- sodium sulfite (stored in chemical cabinets)
- top-loading balance (2 decimal places)
- alpha-amylase (stored in refrigerator)
- 5 mL pipet
- neutral detergent solution (stored in room 113B)
- 2000 mL flask
- acetone (in flammables cabinet below fume hood in room 113B)
- digestion apparatus (ANKOM fiber analyzer)
- analytical balance
- drying oven at 105°C
- zip-loc bags with dessicant pouches (recently recharged at 105°C)
- 3 glass jars
- hazardous waste container
- Close valve on ANKOM fiber analyzer.
- Load filter bags into bag suspender, starting with bottom tray. Place three bags per tray and stack trays on center post with each level rotated 120 degrees. Insert bag suspender into fiber analyzer.
- Weigh out 20 g sodium sulfite and add to fiber analyzer.
- Measure out approximately 2 L neutral detergent solution into flask (this may need to be done 2 x 1 L, due to foam). Add 4 mL alpha-amylase to solution. Pour mixture into fiber analyzer. Place weight on bag suspender, turn on agitation and check liquid level.
- Close lid. Heat to 100°C and agitate for 75 min.
- After 60 min, fill 3 glass jars (to just below neck) with nanopure water. Microwave 1st jar for 11 min.
- After 75 min, turn off heat and agitation. Open valve so solution drains into sink with cold water running.
- Open lid. Close valve. Add 4 mL alpha-amylase to heated rinse water. Pour rinse into fiber analyzer. Agitate for 5 minutes with lid open. Microwave 2nd jar of nanopure water for 11 min.
- Repeat 2nd rinse with 4 mL alpha-amylase.
- For final (3rd) rinse, use hot water only.
- After final rinse is complete, remove bag suspender from fiber analyzer. Gently squeeze bags (4-6 at a time) to remove excess water.
- Place bags in 1 L beaker. Add acetone to cover (~400 mL) and let soak in fume hood for 3-5 min. Gently squeeze bags (4-6 at a time) to remove acetone. Let air-dry on clean tray in fume hood for 1 h (until acetone has evaporated). Pour used acetone into hazardous waste container.
- Place tray in 105°C oven to dry overnight.
- Remove bags from oven and place immediately in zip-loc bags (flatten to remove air) and into dessicator. After cool (~30 min), record NDF bag weight using appropriate analytical balance. Proceed with ADF.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4.
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56.
- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. ISBN:0879697164
- Who has experience with this protocol?
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