RNA isolation / purification Bacteria - Gram negative Haemophilus influenzae

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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ChargeSwitch™ Total RNA Cell Kit

Thermo Fisher Scientific

Upstream tips
Store DNase I at -20°C immediately upon receipt. Do not store DNase I at room temperature.
Protocol tips
Ensure that the sample is fully homogenized before proceeding to 60°C incubation.
After adding Elution Buffer (E7) to the sample, pipet up and down to resuspend the magnetic beads before incubation.
Upstream tips
For lysis/cell disruption check the protocol beforehand to see which reagents/lab materials are needed
Downstream tips
Minimal DNA is left over using this kit, however if DNA is undesired an additional RNase free DNase treatment is warranted.
Upstream tips
Be careful to create an RNase-free working environment
Protocol tips
Always mix the sample tube well after addition of each reagent.
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