Chapter Nineteen - RNA Purification – Precipitation Methods
Section snippets
Theory
RNA dissolves readily in water because both are highly polar substances in nature. In order to remove RNA from water, the charged backbone must be neutralized. This is generally achieved by the addition of monovalent cations in the form of salts (i.e., sodium acetate, ammonium acetate, lithium chloride) and in some cases alcohol. While the salt provides a positive charge to neutralize the phosphate backbone of RNA, the alcohol allows the salt to interact with RNA more effectively by changing
Equipment
Microcentrifuge or floor centrifuge with appropriate rotors
Micropipettors
Micropipettor tips
1.5-ml microcentrifuge tubes
Beakers
1 l Graduated cylinders
Sterile 0.22-μm filter units
50-ml polypropylene conical tubes
Materials
RNA to be precipitated
100% Ethanol
Lithium chloride (LiCl)
Sodium acetate (NaOAc)
Ammonium acetate (NH4OAc)
Glacial acetic acid
Type I grade water (Molecular Biology grade, free of RNases)
Dry ice (optional)
95% Ethanol or Methanol (optional)
Glycogen
Preparation
Prepare RNA by isolating it from cells, polysomes (see Analysis of polysomes from bacteria, or Polysome Profile Analysis - Yeast, or Polysome analysis of mammalian cells or Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae), or by in vitro transcription (see In Vitro Transcription from Plasmid or PCR amplified DNA). Phenol/chloroform extraction is recommended prior to ethanol precipitation.
Duration
Preparation 1 day Protocol 1 h–1 day
Tip
RNAses are a ubiquitous
Overview
Precipitate RNA by incubating with a salt of choice and ethanol. Collect RNA by centrifugation, rinse pellet with 70% ethanol, and dissolve RNA pellet.
Duration
1 h to overnight
- 1.1
Add 0.1 volumes of 7.5 M ammonium acetate or 3 M sodium acetate and mix. Then add 2.5 volumes of 100% ethanol.
- 1.2
Incubate for 25 min in a dry ice/ethanol bath or for 2 h to overnight at − 20 °C.
- 1.3
Pellet RNA at 12 000 × g for 15 min (maximum speed in a microcentrifuge).
- 1.4
Carefully remove the supernatant and wash the RNA pellet (do not try to
Overview
Precipitate RNA using lithium chloride. Lithium chloride does not precipitate tRNA and may not work as well for RNAs under ~ 100 nucleotides or very dilute RNAs. Lithium chloride precipitation removes nucleotides and most proteins.
Duration
1 h
- 2.1
Add an equal volume of 7.5 M lithium chloride.
- 2.2
Incubate in dry ice/ethanol for 15 min or for at least 30 min at − 20 °C.
- 2.3
Pellet RNA at 12 000 × g for 15 min (maximum speed in a microcentrifuge).
- 2.4
Carefully remove the supernatant and wash the RNA pellet (do not try to
References (3)
- et al.
A method for isolation of intact, translationally active ribonucleic acid
DNA
(1983)
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