ChargeSwitch™ Total RNA Cell Kit

RNA isolation / purification Bacteria - Gram negative Haemophilus influenzae

Experiment
RNA isolation / purification Bacteria - Gram negative Haemophilus influenzae
Product
ChargeSwitch™ Total RNA Cell Kit from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Upstream tips
Store DNase I at -20°C immediately upon receipt. Do not store DNase I at room temperature.
Protocol tips
Ensure that the sample is fully homogenized before proceeding to 60°C incubation.
After adding Elution Buffer (E7) to the sample, pipet up and down to resuspend the magnetic beads before incubation.

Publication protocol

Cultures of the wild-type and ΔtoxAvapA mutant were grown in 35 ml of defined media [16] in baffled flasks at 35°C with shaking. Once the OD600 reached ∼0.4, five ml of culture was added to 5 ml of RNAlater Solution (Ambion, Life Technologies, Grand Island, NY USA) and stored at 4°C until processed. The experiment was repeated in triplicate and all repetitions were processed within 48 hours of collection. Total RNA was isolated using the ChargeSwitch Total RNA Cell Kit (Invitrogen, Life Technologies, Grand Island, NY USA) according to the manufacturer's directions, with the modification of a vial change and an extended DNAse incubation step (30 min at 37°C). Following purification, the concentration of the total RNA was determined by a Nanodrop 2000c spectrophotometer (Thermo Scientific Inc., Pittsburgh, PA USA) and aliquots were prepared and stored at −80°C. Prior to cDNA synthesis, PCR using an aliquot of total RNA as the template was performed to confirm that each preparation was free of contaminating genomic DNA.



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Discussion

Discussion

2 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

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Bacteria - Gram negative Haemophilus influenzae using ChargeSwitch™ Total RNA Cell Kit from Thermo Fisher Scientific.

Paper title
The Toxin-Antitoxin Locus Contributes to the Survival of Nontypeable during Infection
Full paper
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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for ChargeSwitch™ Total RNA Cell Kit below.

Download PDF Download manufacturer protocol

Videos

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