Spin media at 500-1,000 x g for 10 min (optional), pre-x g for 10 min, filter with 0.22 µm PES membrane, freeze at -80°C.
{"action": "spin", "output": "", "speed": ["500-1,000 x g"], "time": ["10 min"]}
Thaw 4 ml supernatant on ice, add 4 ml XBP buffer, .
{"action": "add", "output": "", "volume": ["4 ml XBP buffer"], "reagent": ["supernatant"]}
Add sample/XBP mix to exoEasy maxi spin column, centrifuge 1-3 min at 500 x g, discard flow-through. #####%%%%% output "flow-through" are not used
{"action": "add", "output": "flow-through", "reagent": ["sample/XBP mix"], "container": ["exoEasy maxi spin column"]}
Add 10 ml XWP to spin column, centrifuge 5 min at 5,000 x g, transfer column to fresh collection tube. #####%%%%% output "Qiazol" are used in line10
{"action": "centrifuge", "output": "Qiazol", "volume": ["10 ml XWP"], "time": ["5 min"], "speed": ["5,000 x g"], "device": ["spin column"]}
Add 700 µL Qiazol to spin column, centrifuge 5 min at 5,000 x g, spin PLG tubes 30 s at 16,000 x g. 7.
{"action": "add", "output": "", "reagent": ["Qiazol"]}
Add flow-through to PLG tube, vortex 5 s, incubate 5 min at RT.
{"action": "add", "output": "", "reagent": ["flow-through"], "container": ["PLG tube"]}
Add 90 µL chloroform, .
{"action": "add", "output": "", "volume": ["90 \u00b5L chloroform"]}
shake vigorously for 15 s, incubate 2-3 min at RT
{"action": "shake", "output": "", "time": ["15 s"], "temperature": ["RT"]}
Centrifuge 15 min at 12,000 x g, transfer upper aqueous phase to new tube, . #####%%%%% output "upper aqueous phase" are not used
{"action": "transfer", "output": "upper aqueous phase", "container": ["new tube"]}
add 2 volumes 100% ethanol, mix
{"action": "add", "output": "", "volume": ["2 volumes"], "reagent": ["upper aqueous phase"]}
Add mix to MinElute spin column, centrifuge 15 s at 1,000 x g, discard flow-through, repeat until all sample is used.
{"action": "centrifuge", "output": "", "container": ["MinElute spin column"], "time": ["15 s"], "speed": ["1,000"]}
Wash column with 700 µL Buffer RWT, centrifuge 15 s at ≥8,000
{"action": "centrifuge", "output": "", "volume": ["700 \u00b5L"], "time": ["15 s"], "speed": [">=8,000"]}
Wash twice with 500 µL Buffer RPE, centrifuge 15 s at ≥8,000 #####%%%%% output "RNase-free" are used in line28
{"action": "centrifuge", "output": "RNase-free", "volume": ["500 \u00b5L Buffer RPE"], "time": ["15 s"], "speed": [">=8,000"]}
Transfer column to new 2 ml tube, dry membrane by centrifuging at full speed for 5 min, transfer to new 1.5 ml tube, add RNase-free water. #####%%%%% output "RNA" are used in line30
{"action": "add", "output": "RNA", "reagent": ["RNase-free"]}
, centrifuge to elute RNA
{"action": "centrifuge", "output": "", "reagent": ["RNA"], "speed": [""], "time": [""]}