RNA isolation / purification Bacteria - Gram positive Clostridium tetani

The biggest problem in isolating RNA from gram-positive bacteria is the disruption of the cell wall. A lot of protocols employ enzymatic digestion (pretreatment) which may affect gene expression patterns of certain genes. Therefore physical disruption using beads can be a best alternative.

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5 years ago

5 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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Found 2 matching solutions for this experiment

RNeasy Mini Kit + RNeasy Protect Bacteria Mini Kit

RNeasy Mini Kit

Qiagen

RNeasy Protect Bacteria Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
To ensure reliable and reproducable gene expression, minimal media is recommended, though complex media also work with this kit.	To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

TRIzol Reagent

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

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

Manufacturer protocol Publication protocol 1 Relevant paper

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