RNA isolation / purification Bacteria - Gram negative Salmonella typhi

Generally isolating RNA from Gram-negative bacteria is easy, however keeping your working environment clean and RNase free (use RNase inhibitor) is essential. Some common points to keep in mind: a) Use fresh samples for isolation or store them by freezing in RNA stabilizing buffer until use. b) Choose the bacterial input amounts carefully, to ensure buffer volumes are adequate and not to overload the columns.

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1 year ago

1 year ago by 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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Found 3 matching solutions for this experiment

Upstream tips
To ensure reliable and reproducable gene expression, minimal media is recommended, though complex media also work with this kit.
Downstream tips
RNA protect only stabilizes the RNA and lyses the bacteria, for further purification RNeasy mini or midi kits are recommended by the manufacturer.
TRIzol Reagent

Thermo Fisher Scientific

Protocol tips
For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.
TRI Reagentâ„¢ Solution

Thermo Fisher Scientific

Upstream tips
Carefully watch how much TRI reagent should be added to your samples
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