High Pure RNA Isolation Kit
RNA isolation / purification Bacteria - Gram positive Streptococcus pneumoniae
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
Frozen cell pellets were resuspended in 400 μl TE (DEPC) and transferred to the screw-capped tubes containing 0.5 g glass beads, 50 μl 10% SDS and 500 μl phenol:chloroform:isoamyl alcohol (25:24:1). The cells were lysed by using a bead beater. The lysed samples were then centrifuged at 10,000 rpm for 10 min (4 °C). The upper phase was transferred to fresh tubes containing 500 μl chloroform:IAA (24:1) and centrifuged for 5 min at 10,000 rpm (4 °C). The 500 μl of upper phase was again transferred to fresh tubes containing 2 × volumes (1 ml) of lysis/binding buffer provided with high pure RNA isolation kit (Roche international). All other steps including the DNaseI were performed according to the manual provided with high pure RNA isolation kit. The concentration of RNA was checked on a spectrophotometer and quality of RNA was checked using Agilent RNA analysis kit (Agilent technologies).Full paper Login or join for free to view the full paper.
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Discussion
Discussion
4 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.
Download manufacturer protocol
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