Replace this sentence with a brief description of the protocol and its goal.
- 2000 mL flask
- acetone (in flammables cabinet below fume hood in room 113B)
- 5 glass jars
- pH paper
- concentrated sulfuric acid
- top-loading balance (1 decimal place)
- ANKOM Daisy incubator and jars
- analytical balance
- drying oven at 105°C
- zip-loc bags with dessicant pouches (recently recharged at 105°C)
- hazardous waste containers
- Prepare 72% (w/w) sulfuric acid the night before performing ADL. Wear appropriate safety equipment (lab coat, safety glasses, rubber gloves (over-top of blue nitrile gloves). Move balance into fume hood.
- Weigh 440 g nanopure water into 2000 mL flask.
- Weigh 400 g concentrated sulfuric acid into 400 mL beaker. Slowly add to water in flask.
- Repeat sulfuric acid addition 2 more times for a total of 1200 g sulfuric acid. This makes ~1 L 72% sulfuric acid, enough for 2 ADL batches.
- Cover flask with foil and let cool overnight in the fume hood.
- Label 2 Daisy incubator jars with batch number.
- Add 1 batch of bags to each jar (~10-12 bags on each side of divider).
- Split 72% sulfuric acid evenly between two jars (~500 mL in each jar).
- Place lids on jars and put in Daisy incubator.
- Rotate for 3 hours with door to Daisy incubator open.
- After 2 h 45 min, fill 5 glass jars (to just below neck) with nanopure water. Microwave 1st jar for 11 min.
- After 3 h, pour acid into hazardous waste container using funnel.
- Add heated rinse water to jars (split evenly between jars) and rotate jars in Daisy incubator while heating next rinse in microwave.
- Repeat rinses until water is neutral pH. Check this using pH paper and compare to nanopure water for reference. Should require 5 rinses. After 1st rinse, water can be disposed of down drain with running water.
- After final rinse is complete, remove bags from jars (do 1 batch at a time so they do not get mixed together). 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 ADL bag weight using appropriate analytical balance. Proceed with ash content determination.
- ALWAYS add acid to water and work in fume hood when making 72% sulfuric acid
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.
- Who has experience with this protocol?
or instead, discuss this protocol. and