Prepare reagents according to recipes 1. #####%%%%% output "recipes 1" are used in line12
{"action": "prepare", "output": "recipes 1", "reagent": ["reagents"]}
Note that everything is in DEPC water.
{"action": "note", "output": "", "reagent": ["DEPC water"], "device": [""]}
Collect the cells by spinning down without freezing on ice.
{"action": "collect", "output": "", "reagent": ["cells"], "device": ["centrifuge"]}
Re-suspend cells with 1 mL water , quickly spin down at 3,000 x g for 15 sec. #####%%%%% output "AE buffer" are used in line10
{"action": "spin", "output": "AE buffer", "time": ["15 sec"], "reagent": ["cells"], "volume": ["1 mL water"]}
Re-suspend cell pellet in 400 µL of AE buffer at room temperature.
{"action": "suspend", "output": "", "reagent": ["AE buffer"], "volume": ["400 µL"], "temperature": ["room temperature"]}
Add 40 µL 10% SDS (final around 1%) .
{"action": "add", "output": "", "reagent": ["10% SDS"], "volume": ["40 µL"]}
Immediately add 500 µL hot phenol/AE (put in 65 °C for 10 min before use), .
 {"action": "add", "output": "", "reagent": ["hot phenol/AE"], "volume": ["500 µL"], "temperature": ["65 °C"]}
vortex vigorously for 1 min
{"action": "sort", "output": "", "reagent": ["phenol/AE"], "time": ["1 min"]}
Incubate at 65 °C for 5 min.
{"action": "incubate", "output": "", "temperature": ["65 \u00b0C"], "time": ["5 min"]}
Immediately freeze by dumping into liquid nitrogen (labels won’t get off).
{"action": "suspend", "output": "", "reagent": ["liquid nitrogen"]}
Wait to thaw at RT (put in 30 °C to thaw .
{"action": "see", "output": "", "reagent": ["cells"], "temperature": ["30 °C"]}
may crack the tube)

Transfer around 400 μL supernatant to a new eppendorf tube. #####%%%%% output "supernatant" are used in line32
{"action": "transfer", "output": "supernatant", "volume": ["400 μL"]}
Recycle the lower phenol fraction carefully following the chemical safety protocol in your laboratory.

Add equal volume (400 μL) phenol: CHCl3/AE-Na.
{"action": "add", "output": "", "volume": ["400 µL"], "reagent": ["phenol: CHCl3/AE-Na"]}
Spin down at 20,000 x g for 5 min in a standard laboratory microfuge. #####%%%%% output "CHCl3" are used in line34
{"action": "spin", "output": "CHCl3", "force": ["20,000 x g"], "time": ["5 min"], "device": ["a standard laboratory microfuge"], "reagent": ["supernatant"]}
Add CHCl3: isoamyl alcohol (24:1).
{"action": "add", "output": "", "reagent": ["CHCl3"], "container": ["isoamyl alcohol (24:1)"]}
Transfer aqueous supernatant to a fresh 1.5 mL microfuge tube. #####%%%%% output "aqueous supernatant" are used in line38
{"action": "transfer", "output": "aqueous supernatant", "container": ["a fresh 1.5 mL microfuge tube"]}
If white cloudy precipitate is observed between the aqueous phase , repeat steps 17-18.
{"action": "repeat", "output": "", "container": ["the aqueous phase"], "reagent": ["aqueous supernatant"]}
Add 1/10 volume of 3 M NaOAc (pH 5) .
{"action": "add", "output": "", "reagent": ["3 M NaOAc (pH 5)"], "volume": ["1/10 volume"]}
Add 2.5 volumes of ethanol.
{"action": "add", "output": "", "volume": ["ethanol"]}
Place at -20 °C for at least 30 min.
{"action": "place", "output": "", "time": ["at least 30 min"], "temperature": ["-20 °C"]}
Spin down in the microfuge at 20,000 x g, 15 min at 4 °C.
{"action": "spin", "output": "", "device": ["the microfuge"], "force": ["20,000 x g"], "time": ["15 min"], "temperature": ["4 °C"]}
Add ice-cold 75% EtOH, place at 4 °C for around 10 min. #####%%%%% output "the liquid droplets" are used in line50
{"action": "add", "output": "the liquid droplets", "reagent": ["ice-cold 75% EtOH"], "time": ["around 10 min"], "temperature": ["4 °C"]}
Suck out the liquid droplets in the tube.
{"action": "pour", "output": "", "reagent": ["the liquid droplets"], "container": ["the tube"]}
Add 30-50 µL ddH2O (DEPC) immediately after it becomes clear. #####%%%%% output "RNA" are used in line54
{"action": "add", "output": "RNA", "volume": ["30-50 µL"], "container": ["\u00b5L ddH2O (DEPC)"]}
If RNA pellet is over-dry, dissolve RNA at 37 °C for 30 min.
{"action": "dissolve", "output": "", "reagent": ["RNA"], "time": ["30 min"], "temperature": ["37 °C"]}