RNA isolation / purification Bacteria - Gram negative Vibro parahaemolyticus

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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Protocol tips
Total RNA was extracted from the tissues according to the manufacturer’s instructions (Spin-column, BioTeke, Beijing, China). First-strand cDNA was obtained using PrimeScript First-strand cDNA synthesis kit with Oligo dT Primer from Takara (Dalian, China). The cDNA mixture was diluted 10 times with PCR-grade water and then stored at −80 °C for qRT-PCR analysis.
Downstream tips
This product stabilizes bacterial RNA and aids in bacterial lysis, TRIzol or a similar product is required for further processing.
Upstream tips
Be careful to create an RNase-free working environment
Protocol tips
Always mix the sample tube well after addition of each reagent.
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.
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