RNeasy Mini Kit

RNA isolation / purification Bacteria - Gram positive Clostridium tetani

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

Protocol tips

Upstream tips
To ensure reliable and reproducable gene expression, minimal media is recommended, though complex media also work with this kit.
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

RNA protect only stabilizes the RNA and lyses the bacteria, for further purification RNeasy mini or midi kits are recommended by the manufacturer.

Publication protocol

RNA extraction was performed using the RNeasy Mini Kit (Qiagen). Cells were harvested and lysed according to the Appendix C protocol of the RNAprotect Bacteria Reagent Handbook (Second Edition, December 2005). Protocol 7 was used for the purification of either total RNA or DNA from bacterial lysate, either with or without optional on-column DNase digestion. Integrity of RNA preparations were visualized on a 1.2% agarose gel and purity and concentration were assessed by OD260/OD280.

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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

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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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