Jul 07, 2020
Phenol-chloroform extraction and ethanol precipitation of RNA
- 1W. Szafer Institute of Botany, Polish Academy of Sciences
Protocol Citation: Tomasz Suchan 2020. Phenol-chloroform extraction and ethanol precipitation of RNA. protocols.io https://dx.doi.org/10.17504/protocols.io.rzvd766
Manuscript citation:
New England Biolabs HiScribeTM T7 High Yield RNA Synthesis Kit Instruction Manual
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 26, 2018
Last Modified: July 07, 2020
Protocol Integer ID: 14101
Abstract
Based on the protocol form NEB HiScribeTM T7 High Yield RNA Synthesis Kit
Materials
MATERIALS
Ethanol 100%
Ethanol 70% [Note: freshly prepared]
nuclease free water
sodium acetate 3M solution
EDTA 0.1 mM
Adjust the reaction volume to 180 μl by adding 160 μl nuclease-free water. Add 20 μl of 3 M sodium acetate, pH 5.2 or 20 μl of 5 M ammonium acetate (1/10 of the total volume), mix thoroughly.
Extract with an equal volume (200 μl) of 1:1 phenol/chloroform mixture. Vortex and centrufugate at maximum speed for 5 min.
Collect the aqueous (upper) phase and transfer to a new tube.
Repeat steps 2 and 3.
Precipitate the RNA by adding 2 volumes of -20°C 100% ethanol. Incubate at –20°C or on dry ice for at least 30 minutes.
Collect the pellet by centrifugation at 4°C for 30 min.
Remove the supernatant and rinse the pellet with 500 μl of cold 70% ethanol.
Quick spin and collect the last drop of ethanol.
Dry the sample.
Resuspend the RNA in 50 μl of 0.1 mM EDTA. Store the RNA at –20°C or below.