ISOLATE II RNA Micro Kit

RNA isolation / purification Bacteria - Gram negative Vibro cholerae

Experiment
RNA isolation / purification Bacteria - Gram negative Vibro cholerae
Product
ISOLATE II RNA Micro Kit from Bioline
Manufacturer
Bioline

Protocol tips

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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Discussion

Discussion

5 years ago

Author: Paul G. Macon United States

Some help with RNA isolation using Trizol

Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?

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Papers

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Paper title
Identification and Characterization of VpsR and VpsT Binding Sites in
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Manufacturer protocol

Download the product protocol from Bioline for ISOLATE II RNA Micro Kit below.

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