RNA isolation / purification Bacteria - Gram negative Bordetella pertussis

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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2 years ago

2 years 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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Upstream tips
"The MagNA Pure LC 2.0 Instrument, together with its software is required for analysing the yields of this kit.
Do not use more sample than this kit is designed to handle.
Do not allow Wash Buffer I or the Lysis/Binding Buffer to mix with sodium hypochlorite (bleach) solution. This mixture can produce a highly toxic gas"
NucleoSpin® RNA

Macherey Nagel

Upstream tips
Aliquot rDNase and store at -20 °C.
Protocol tips
Dry the columns very well after the ethanol wash by adding an additional 2' >10000 rpm centrifuge with no buffer.
Protocol tips
- To capture a higher amount of mRNA, modify the amount of lysis buffer :ethanol (from 1:1 to 1:1.5) during the binding step.
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