ISOLATE II RNA Micro Kit

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	Total RNA was isolated from V. cholerae cells grown aerobically to mid-exponential phase (optical density at 600 nm [OD600] = 0.3 to 0.4) in LB medium at 30°C. Briefly, overnight-grown V. cholerae cultures were diluted 1:200 in fresh LB medium and were grown aerobically at 30°C with shaking at 200 rpm to an OD600 of 0.3 to 0.4. The cultures were reinoculated (1:200) into fresh LB medium until the OD600 reached 0.3 to 0.4. Aliquots (2 ml) of the cultures were collected and centrifuged for 2 min at room temperature. Cell pellets were immediately resuspended in 1 ml of TRIzol reagent (Invitrogen, Grand Island, NY) and stored at −70°C. Total RNA was isolated according to the manufacturer's instructions. To remove contaminating DNA, total RNA was incubated with a Turbo DNA-free kit (Thermo Scientific, Grand Island, NY) and concentrated using an Isolate II RNA micro-cleanup kit (Bioline, Taunton, MA).

Protocol tips

Total RNA was isolated from V. cholerae cells grown aerobically to mid-exponential phase (optical density at 600 nm [OD600] = 0.3 to 0.4) in LB medium at 30°C. Briefly, overnight-grown V. cholerae cultures were diluted 1:200 in fresh LB medium and were grown aerobically at 30°C with shaking at 200 rpm to an OD600 of 0.3 to 0.4. The cultures were reinoculated (1:200) into fresh LB medium until the OD600 reached 0.3 to 0.4. Aliquots (2 ml) of the cultures were collected and centrifuged for 2 min at room temperature. Cell pellets were immediately resuspended in 1 ml of TRIzol reagent (Invitrogen, Grand Island, NY) and stored at −70°C. Total RNA was isolated according to the manufacturer's instructions. To remove contaminating DNA, total RNA was incubated with a Turbo DNA-free kit (Thermo Scientific, Grand Island, NY) and concentrated using an Isolate II RNA micro-cleanup kit (Bioline, Taunton, MA).

Publication protocol

Total RNA was isolated from V. cholerae cells grown aerobically to mid-exponential phase (optical density at 600 nm [OD600] = 0.3 to 0.4) in LB medium at 30°C. Briefly, overnight-grown V. cholerae cultures were diluted 1:200 in fresh LB medium and were grown aerobically at 30°C with shaking at 200 rpm to an OD600 of 0.3 to 0.4. The cultures were reinoculated (1:200) into fresh LB medium until the OD600 reached 0.3 to 0.4. Aliquots (2 ml) of the cultures were collected and centrifuged for 2 min at room temperature. Cell pellets were immediately resuspended in 1 ml of TRIzol reagent (Invitrogen, Grand Island, NY) and stored at −70°C. Total RNA was isolated according to the manufacturer's instructions. To remove contaminating DNA, total RNA was incubated with a Turbo DNA-free kit (Thermo Scientific, Grand Island, NY) and concentrated using an Isolate II RNA micro-cleanup kit (Bioline, Taunton, MA).



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