High Pure RNA Isolation Kit
RNA isolation / purification Bacteria - Gram positive Lactobacillus amylovorus
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).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?
Papers
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Manufacturer protocol
Download the product protocol from Roche Lifesciences for High Pure RNA Isolation Kit below.
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