TRIzol Reagent
RNA isolation / purification Bacteria - Gram positive Clostridum botulinum
Protocol tips
| Protocol tips |
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| For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it. |
Publication protocol
Total RNA was extracted from BoNT/A-treated cells using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified using RNeasy columns (Qiagen, Valencia, CA) according to the manufacturers’ protocols. After contaminating DNA was degraded with 20 U RNase-free DNase (Promega), the concentration and purity of RNA samples were evaluated with denaturing gel electrophoresis and by measuring the absorbance at 260 and 280 nm with an Agilent 2100 analyzer (Agilent Technologies, Palo Alto, CA). RNA preparations for microarray were independently performed in triplicate under the same conditions.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
5 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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