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Phenol-based Extraction of RNA from Chlamydomonas reinhardtii

Emanuel Sanz-Luque Emanuel Sanz-Luque
AM Amaury de Montaigu

Published: Jul 5, 2018 DOI: 10.21769/BioProtoc.2910 Views: 6031

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 search DNase treatment search Phenol extraction search Chlamydomonas search

Materials and Reagents

  1. Gloves
  2. Pipette tips
  3. 2 ml microcentrifuge tubes
  4. Liquid nitrogen (N2)
  5. SDS 20%
  6. Water-saturated Phenol (pH 4.5) (Amresco, catalog number: 0981-400ML )
  7. Chloroform:Isoamyl Alcohol (24:1) (Amresco, catalog number: X205-450ML )
  8. Chloroform (Sigma-Aldrich, catalog number: 372978 ) (store at 4 °C)
  9. 8 M Lithium Chloride (LiCl, store at 4 °C)
  10. 70% and 100% ethanol (store at 4 °C)
  11. Nuclease-free water
  12. TURBOTM DNase (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2238 )
  13. 3 M Sodium Acetate
  14. Agarose
  15. Extraction Buffer (see Recipes)
  16. Phenol (pH 4.5):Chloroform:Isoamyl Alcohol (see Recipes) (store at 4 °C)

Equipment

  1. Pipettes
  2. Autoclave
  3. Centrifuge
  4. Nanodrop (Thermo Fisher Scientific, model: NanoDropTM 1000 , catalog number: ND-1000)

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant molecular biology > RNA > RNA extraction

Microbiology > Microbial genetics > RNA > RNA extraction

Molecular Biology > RNA > RNA extraction

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