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Phenol-based Extraction of RNA from Chlamydomonas reinhardtii
Published: Jul 5, 2018 DOI: 10.21769/BioProtoc.2910 Views: 6003
Edited by: Adam Idoine Reviewed by: Antoine Danon
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Abstract
RNA extraction is a basic procedure in molecular biology and a wide variety of commercial kits are available. Some of these kits have been successfully used in Chlamydomonas. However, in some cases RNA quality and quantity may be dramatically reduced depending on the strains and/or the conditions where cells were grown or treated. Phenol-based protocols are the most robust methods to obtain both high quantity and quality RNA from any strain of this alga grown in any condition. Here, we describe an easy and cheap protocol using phenol but avoiding the acute toxicity of guanidine isothiocyanate present in the commercial phenol-based mixtures.
Keywords: RNA
DNase treatment
Phenol extraction
Chlamydomonas
Materials and Reagents
- Gloves
- Pipette tips
- 2 ml microcentrifuge tubes
- Liquid nitrogen (N2)
- SDS 20%
- Water-saturated Phenol (pH 4.5) (Amresco, catalog number: 0981-400ML )
- Chloroform:Isoamyl Alcohol (24:1) (Amresco, catalog number: X205-450ML )
- Chloroform (Sigma-Aldrich, catalog number: 372978 ) (store at 4 °C)
- 8 M Lithium Chloride (LiCl, store at 4 °C)
- 70% and 100% ethanol (store at 4 °C)
- Nuclease-free water
- TURBOTM DNase (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2238 )
- 3 M Sodium Acetate
- Agarose
- Extraction Buffer (see Recipes)
- Phenol (pH 4.5):Chloroform:Isoamyl Alcohol (see Recipes) (store at 4 °C)
Equipment
- Pipettes
- Autoclave
- Centrifuge
- Nanodrop (Thermo Fisher Scientific, model: NanoDropTM 1000 , catalog number: ND-1000)
Procedure
Category
Plant Science > Plant molecular biology > RNA
Microbiology > Microbial genetics > RNA
Molecular Biology > RNA > RNA extraction
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