TRIzol™ LS Reagent
RNA isolation / purification Bacteria - Gram positive Listeria monocytogens
Protocol tips
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| TRIzol LS reagent is intended for liquid samples, using solid samples will decrease RNA yield |
Publication protocol
For RNA isolation, pellets were resuspended in 750 µl TRIzol Reagent (Life Technologies). Cells were disrupted using beadbeating in Lysing Matrix A tubes (MP Biomedicals) with a FastPrep FP120 instrument (MP Biomedicals) with the following parameters: three times 45s at speed 5.5 at 4°C. RNA isolation was performed according to the manufacturer’s instructions. Concentration of nucleic acids was measured with a Nanodrop instrument (Shimadzu BioSpec-nano) and remaining DNA was removed using the Turbo DNA-free Kit (Life Technologies) according to manufacturer’s instructions. The absence of DNA was confirmed by performing a PCR using qacH fwd and rev primers. PCR conditions were the same as indicated above, except that 40 cycles were used.Full paper Login or join for free to view the full paper.
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Discussion
Discussion
5 years ago
Author:
Paul G. Macon
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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Manufacturer protocol
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