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(New page: '''Neutral Detergent Fiber''' '''Materials:''' *sodium sulfite (stored in chemical cabinets) *top-loading balance (2 decimal places) *spatula *alpha-amylase (stored in refrigerator) *5 mL...)
 
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'''Neutral Detergent Fiber'''
==Overview==
 
Type something here
 
==Materials==


'''Materials:'''
*sodium sulfite (stored in chemical cabinets)
*sodium sulfite (stored in chemical cabinets)
*top-loading balance (2 decimal places)
*top-loading balance (2 decimal places)
Line 9: Line 12:
*neutral detergent solution (stored in room 113B)
*neutral detergent solution (stored in room 113B)
*2000 mL flask
*2000 mL flask
*acetone (in flammables cabinet below fume hood in room 113B)
*acetone (in flammables cabinet below fume hood in room 113B)
*digestion apparatus (ANKOM fiber analyzer)
*digestion apparatus (ANKOM fiber analyzer)
*analytical balance
*analytical balance
*drying oven at 105oC
*drying oven at 105°C
*dessicator
*dessicator
*zip-loc bags with dessicant pouches (recently recharged at 105oC)
*zip-loc bags with dessicant pouches (recently recharged at 105°C)
*3 glass jars
*3 glass jars
*microwave
*microwave
*hazardous waste container
*hazardous waste container


'''Procedure'''
==Procedure==
 
#Close valve on ANKOM fiber analyzer.
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.
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.
#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.
Weigh out 20 g sodium sulfite and add to fiber analyzer.
#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.   
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.
#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.
Close lid.  Heat to 100oC and agitate for 75 min.
#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. 
After 60 min, fill 3 glass jars (to just below neck) with nanopure water.  Microwave 1st jar for 11 min.
#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.
After 75 min, turn off heat and agitation.  Open valve so solution drains into sink with cold water running.   
#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.
 
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.
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
#List troubleshooting tips here. 
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Anecdotal observations that might be of use to others can also be posted here.


After final rinse is complete, remove bag suspender from fiber analyzer.  Gently squeeze bags (4-6 at a time) to remove excess water.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.


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.
==References==
'''Relevant papers and books'''
<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
<biblio>
#Goldbeter-PNAS-1981 pmid=6947258
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
</biblio>


Place tray in 105oC oven to dry overnight.
==Contact==
*Who has experience with this protocol?


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.
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].

Latest revision as of 10:27, 3 February 2010

Overview

Type something here

Materials

  • sodium sulfite (stored in chemical cabinets)
  • top-loading balance (2 decimal places)
  • spatula
  • 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
  • dessicator
  • zip-loc bags with dessicant pouches (recently recharged at 105°C)
  • 3 glass jars
  • microwave
  • hazardous waste container

Procedure

  1. Close valve on ANKOM fiber analyzer.
  2. 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.
  3. Weigh out 20 g sodium sulfite and add to fiber analyzer.
  4. 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.
  5. Close lid. Heat to 100°C and agitate for 75 min.
  6. After 60 min, fill 3 glass jars (to just below neck) with nanopure water. Microwave 1st jar for 11 min.
  7. After 75 min, turn off heat and agitation. Open valve so solution drains into sink with cold water running.
  8. 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.
  9. Repeat 2nd rinse with 4 mL alpha-amylase.
  10. For final (3rd) rinse, use hot water only.
  11. After final rinse is complete, remove bag suspender from fiber analyzer. Gently squeeze bags (4-6 at a time) to remove excess water.
  12. 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.
  13. Place tray in 105°C oven to dry overnight.
  14. 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.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers and books

  1. 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. DOI:10.1073/pnas.78.11.6840 | PubMed ID:6947258 | HubMed [Goldbeter-PNAS-1981]
  2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 | PubMed ID:13718526 | HubMed [Jacob-JMB-1961]
  3. ISBN:0879697164 [Ptashne-Genetic-Switch]

All Medline abstracts: PubMed | HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.

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