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Isolation of Genomic DNA from Chlamydomonas reinhardtii

Tyler M Wittkopp Tyler M Wittkopp

Published: May 5, 2018 DOI: 10.21769/BioProtoc.2838 Views: 13574

Edited by: Adam Idoine Reviewed by: Yunbing Ma

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Abstract

Chlamydomonas reinhardtii is a soil-dwelling eukaryotic green alga that is widely used as a laboratory model organism for research on photosynthesis, ciliary biology, lipid metabolism and many other aspects of cell biology and physiology. With sequenced nuclear, chloroplast and mitochondrial genomes, Chlamydomonas is also an excellent organism for genetics and genomics research. This protocol describes the isolation of genomic DNA from Chlamydomonas using a standard phenol:chloroform extraction method followed by ethanol precipitation. The protocol requires minimal lab materials, takes approximately 4 h to complete, and can also be used for isolation of genomic DNA from other eukaryotic green algae.

Keywords: Algae search Genomic DNA search Phenol/chloroform extraction search Nucleic acid search DNA purification search

Background

Isolating nucleic acid is a critical first step for cloning and sequencing genetic material and provides the basis for diverse molecular biological studies ranging from gene expression to gene evolution. A number of protocols exist for isolating DNA from algae (Weeks et al., 1986; Fawley and Fawley, 2004; HwangBo et al., 2010). Generally, cells are pelleted by centrifugation and lysed in buffer containing detergents such as SDS to solubilize membranes. This is followed by at least one extraction in phenol:chloroform and at least one extraction in chloroform. In some cases, an RNase treatment step is performed to degrade RNA. DNA is then precipitated by addition of ethanol or isopropanol and incubating on ice or in the freezer. After pelleting and washing the precipitated DNA, it is usually resuspended in water or buffer (e.g., Tris-EDTA) and quantified spectrophotometrically at 260 nm.

The protocol described herein makes use of two phenol/chloroform/isoamyl alcohol extractions and two chloroform/isoamyl extractions, with an RNase treatment step in between. The addition of isoamyl alcohol to the organic solvents prevents foaming and stabilizes the interphase, which contains a high concentration of coagulated proteins. Importantly, this protocol results in high quality genomic DNA that is suitable for downstream applications such as cloning and sequencing.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml microcentrifuge tubes
  3. 50 ml centrifuge tubes
  4. Kimwipe
  5. RNase A (50 mg/ml) (Sigma-Aldrich, catalog number: R6513 )
  6. Phenol/chloroform/isoamyl alcohol pH 8.0 (25:24:1) (Sigma-Aldrich, catalog number: P2069 )
    Notes:
    1. Separates into two phases–use the bottom organic phase.
    2. Use caution when handling this solution; work in a chemical hood and wear gloves, eye protection.
  7. Chloroform/isoamyl alcohol (24:1) (Sigma-Aldrich, catalog number: 25666 )
    Note: Use caution when handling this solution; work in a chemical hood and wear gloves, eye protection.
  8. Absolute ethanol (Sigma-Aldrich, catalog number: 792780 )
  9. 70% ethanol
  10. Nuclease-free water
  11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  12. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
  13. Ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA·Na2·2H2O) (Sigma-Aldrich, catalog number: E5134 )
  14. Ammonium molybdate tetrahydrate [(NH4)6Mo7O24·7H2O] (Sigma-Aldrich, catalog number: A7302 )
  15. Sodium selenite (Na2SeO3) (Sigma-Aldrich, catalog number: S5261 )
  16. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z4750 )
  17. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: M3634 )
  18. Iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma-Aldrich, catalog number: F2877 )
  19. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
  20. Copper(II) chloride dihydrate (CuCl2·2H2O) (Sigma-Aldrich, catalog number: C3279 )
  21. Tris base (Biopioneer, catalog number: C0060 )
  22. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
  23. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3306 )
  24. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
  25. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
  26. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  27. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  28. Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
  29. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
  30. DNA Extraction buffer (see Recipes)
  31. Micronutrient stock solutions (see Recipes)
  32. Stock solutions (see Recipes)
  33. Tris-Acetate-Phosphate (TAP) growth medium (see Recipes)

Equipment

  1. 250 ml culture flasks (e.g., Corning, PYREX®, catalog number: 70980-250 )
  2. Pipettes
  3. Hemacytometer (e.g., Sigma-Aldrich, catalog number: Z359629 ) or automated cell counter (e.g., Bio-Rad Laboratories, model: TC20TM )
  4. Basic light microscope (e.g., Amscope, catalog number: B100B-MS )
  5. Vortex mixer (e.g., Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  6. Centrifuge (e.g., Eppendorf, model: 5810 R ) and microcentrifuge (e.g., Eppendorf, model: 5424 )
  7. Freezer
  8. Nano-Drop 1000/2000 spectrophotometer or Qubit 2.0 fluorometer
  9. Optional: PCR thermal cycler
  10. Autoclave

Procedure

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

Category

Plant Science > Phycology > DNA > Extraction

Plant Science > Plant molecular biology > DNA > DNA extraction

Molecular Biology > DNA > DNA extraction

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