Grow an overnight 5.0 mL LB culture of the strains to be evaluated. #####%%%%% output "pellet" are used in line4
{"action": "grow", "output": "pellet", "reagent": ["the strains"]}
Re-suspend pellet in 300 mL cold Sodium Acetate (0.3M, pH 4.5), 10 mM EDTA. #####%%%%% output "cells" are used in line6
{"action": "suspend", "output": "cells", "reagent": ["pellet"]}
Add cells to 0.3 g of glass beads , then transfer to –20 °C.
{"action": "transfer", "output": "", "reagent": ["cells"], "container": ["glass beads"], "temperature": ["–20 °C"]}
snap freeze in ethanol dry ice bath
{"action": "grab", "output": "", "reagent": ["ethanol dry ice bath"]}
Hold samples here until all cultures are at this point.
{"action": "hold", "output": "", "device": [""]}
Add 300 mL phenol:chloroform (pH 4.7) to the samples.
{"action": "add", "output": "", "volume": ["300 mL phenol:chloroform (pH 4.7)"], "container": ["the samples"]}
Vortex eppendorf tubes 4-5 times for 30-second bursts with incubation on ice in between.
{"action": "vortex", "output": "", "device": ["eppendorf tubes"], "temperature": ["ice"]}
Transfer the top layer (aqueous phase) to new tubes . #####%%%%% output "the top layer (aqueous phase)" are used in line22
{"action": "transfer", "output": "the top layer (aqueous phase)", "container": ["new tubes"]}
repeat the phenol extraction by adding 300 µL phenol:chloroform (pH 4.7), spinning for 15 minutes at 4 °C, at maximum speed, #####%%%%% output "the RNA" are used in line20
{"action": "repeat", "output": "the RNA", "reagent": ["phenol extraction", "phenol:chloroform (pH 4.7)"], "time": ["15 minutes"], "temperature": ["4 °C"]}
Precipitate the RNA by adding 3 volumes of cold 100% ethanol at maximum speed for 25 minutes at 4 °C. #####%%%%% output "the RNA pellet" are used in line22
{"action": "precipitate", "output": "the RNA pellet", "reagent": ["the RNA", "cold 100% ethanol"], "time": ["25 minutes"], "temperature": ["4 °C"]}
Re-suspend the RNA pellet in 60 µL cold sodium acetate (0.3 M, pH 4.5).
{"action": "suspend", "output": "", "reagent": ["the RNA pellet", "cold sodium acetate (0.3 M, pH 4.5)", "the top layer (aqueous phase)"], "volume": ["60 \u00b5L"], "concentration": [""]}
Precipitate the RNA by adding 400 µL 100% ethanol at maximum speed for 25 minutes at 4 °C.
{"action": "precipitate", "output": "", "volume": ["400 \u00b5L 100% ethanol"], "temperature": ["4 \u00b0C"]}
Air dry the pellet while on ice. #####%%%%% output "the pellet" are used in line28
{"action": "dry", "output": "the pellet", "temperature": ["ice"], "device": [""]}
Resuspend the pellet in -50 µL cold sodium acetate (10 mM, pH 4.5). #####%%%%% output "the RNA" are used in line30
{"action": "resuspend", "output": "the RNA", "container": ["the pellet"], "volume": ["50 µL"], "reagent": ["the pellet"]}
Quantitate RNA by spectrophotometry (A260) analyze the integrity of the RNA by running a 2.0% agarose gel. #####%%%%% output "spectrophotometry (A260)" are not used
{"action": "evaluate", "output": "spectrophotometry (A260)", "reagent": ["the RNA"]}