RNeasy Plus Mini Kit
RNA isolation / purification Bacteria - Gram negative Escherichia coli
Protocol tips
| Downstream tips |
|---|
| Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60C. Use water to elute the RNA that is warmed to ~60C. |
Publication protocol
Bacterial cells were harvested from 2 ml cultures at the logarithmic phase (OD600≈0.8). The cultures were grown in Dulbecco’s Modified Eagle Medium (DMEM) medium (Gibco) or tryptone water containing 1.5% Bacto Tryptone (Difco) and 0.5% sodium chloride at 37°C. Total RNA was extracted using the RNAprotect Bacteria Reagent and RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. The RNA samples were treated with DNase and purified using the RNeasy MiniElute Cleanup kit (Qiagen). RT-PCR reactions were carried out using 1 µg of purified RNA and the SuperScript II One Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen) following the manufacturer’s instructions.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
4 years ago
Author:
Paul G. Macon
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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Manufacturer protocol
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