RNeasy Mini Kit

RNA isolation / purification Bacteria - Gram positive Bacillus anthracis

Experiment
RNA isolation / purification Bacteria - Gram positive Bacillus anthracis
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Protocol tips
To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
"Include DNAse treatment for 15-20min.

Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C.

Use water to elute the RNA that is warmed to ~60`C"

Publication protocol

Overnight cultures of strains tested were incubated in LB medium and grown to mid-log phase (OD600 = 0.6) at 37°C with shaking. The cells were harvested by centrifugation, and total RNA was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The primers used for each target are listed in Table ​Table1.1. cDNA was synthesized from 1 μg RNA using Superscript III reverse transcriptase (Invitrogen) and random hexamers following the manufacturer's protocol. The cDNA was diluted 5-fold and was amplified by qPCR. The gene expression levels as indicated by CT values were compared among the strains tested. 16S rRNA was used as a reference gene for normalization. The qPCR was repeated three times.

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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

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Bacteria - Gram positive Bacillus anthracis using RNeasy Mini Kit from Qiagen.

Paper title
The Gene of pXO1 Is Involved in Capsule Biosynthesis of Pasteur II Strain
Full paper
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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Mini Kit below.

Download PDF Download manufacturer protocol

Videos

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