High Pure RNA Isolation Kit

RNA isolation / purification Bacteria - Gram positive Lactobacillus amylovorus

Experiment
RNA isolation / purification Bacteria - Gram positive Lactobacillus amylovorus
Product
High Pure RNA Isolation Kit from Roche Lifesciences
Manufacturer
Roche Lifesciences

Protocol tips

Upstream tips
Be careful to create an RNase-free working environment
Protocol tips
Always mix the sample tube well after addition of each reagent.

Publication protocol

Total RNA was isolated according to the protocol for Gram-positive bacteria outlined by the Roche High Pure Isolation kit (Roche, Indiana, USA), but with minor modifications. The lysozyme step was shortened and the concentration was increased to 100 mg/ml. Additionally, this step was also merged with a bead-beating step to ensure complete cell lysis; the cells were incubated for 60 min at 37°C while shaking at 1400 rpm in a 2 ml stock tube containing 0.1 mm zirconia beads in an Eppendorf Thermomixer. Contaminating DNA was removed with the Turbo DNA-free kit (Invitrogen, Dun Laoghaire, Ireland). The total RNA was ribo-depleted using the Gram-Positive Bacteria Ribo-Zero™ Magnetic Kit (Cambio Ltd., Cambridge, UK) and cleaned using the RNA Clean & Concentrator™-5 (Cambridge Biosciences, Cambridge, UK). The mRNA fragmentation, random-primed cDNA synthesis, adapter ligation, adapter-specific PCR amplification and pooling of the six tagged libraries into one pool were carried out by GATC Biotech (Konstanz, Germany).

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Reviews

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Discussion

Discussion

5 years ago

Author: 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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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Bacteria - Gram positive Lactobacillus amylovorus using High Pure RNA Isolation Kit from Roche Lifesciences.

Paper title
strains cluster according to their mammalian gut source
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Manufacturer protocol

Download the product protocol from Roche Lifesciences for High Pure RNA Isolation Kit below.

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Videos

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