Purification and Concentration of Nucleic Acids

Dennis H. Dowhan1

1 University of Queensland, Brisbane, Queensland, Australia
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 5.2
DOI:  10.1002/9780470089941.et0502s06
Online Posting Date:  September, 2012
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Abstract

The purification and concentration of nucleic acids have become routine procedures in most biology and molecular biology laboratories. This unit covers the basic principles and procedures for the isolation, purification, and manipulation of solutions of DNA or RNA. The basic DNA protocol, using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations ≤1 mg/ml. Purification of DNA using commercially available silica membrane spin columns is presented as an alternate protocol. Isolation and purification of RNA from mammalian cells or tissues is also examined. Use of the protein denaturant guanidine thiocyanate and water‐saturated phenol, followed by concentration by isopropanol precipitation, for producing small samples of RNA, is illustrated in the basic RNA protocol. Curr. Protoc. Essential Lab. Tech. 6:5.2.1‐5.2.21. © 2012 by John Wiley & Sons, Inc.

Keywords: DNA; RNA; phenol; chloroform; guanidine thiocyanate; concentration; precipitation; extraction; ethanol; isopropanol

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA
  • Alternate Protocol 1: Purification of Plasmid DNA Using Silica Membrane Spin Columns
  • Support Protocol 1: Precipitation of DNA Using Isopropanol
  • Support Protocol 2: Concentration of DNA Using Butanol
  • Basic Protocol 2: Single‐Step RNA Isolation from Cultured Cells or Tissues
  • Alternate Protocol 2: Purification and Concentration of RNA and Dilute Solutions of DNA
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Acknowledgments
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA

  Materials
  • 1 mg/ml DNA to be purified (e.g., see unit 4.2)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 (unit 3.3)
  • 100% ethanol, ice cold
  • Crushed dry ice
  • 70% ethanol, room temperature
  • TE buffer, pH 8.0 (unit 3.3)
  • 1.5 ml‐polypropylene microcentrifuge tubes
  • Microcentrifuge
  • 200‐µl pipet tip and pipettor
  • Desiccator or Speedvac evaporator (Savant)

Alternate Protocol 1: Purification of Plasmid DNA Using Silica Membrane Spin Columns

  Materials
  • 1 × 109 cell/ml bacterial culture containing DNA of interest or 0.1 to 1 mg/ml DNA to be purified (5 to 10 µg DNA total); see unit 4.2
  • 6 M sodium iodide (NaI) solution (filter through filter paper, store up to 3 months in the dark at 4°C)
  • Resuspension buffer (see recipe)
  • Lysis solution: 0.2 M NaOH/1.0% (w/v) SDS (store indefinitely at room temperature)
  • Neutralization/binding solution (see recipe)
  • Wash buffer (see recipe)
  • TE buffer, pH 8.5; (unit 3.3) or nuclease‐free H 2O
  • Silica membrane spin columns (e.g., Qiagen, Promega, Invitrogen, Novagen)
  • 1.5‐ml microcentrifuge tubes

Support Protocol 1: Precipitation of DNA Using Isopropanol

  • sec‐Butanol
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol
  • Polypropylene tube

Support Protocol 2: Concentration of DNA Using Butanol

  Materials
  • Denaturing solution (see recipe)
  • 2 M sodium acetate, pH 4 (see recipe)
  • Phenol, water‐saturated (see recipe)
  • 49:1 (v/v) chloroform/isoamyl alcohol or bromochloropropane
  • 100% isopropanol
  • 75% ethanol (prepared with DEPC‐treated water)
  • DEPC‐treated water or freshly deionized formamide (see recipes)
  • Glass Teflon homogenizer
  • 5‐ml polypropylene centrifuge tube
  • Sorvall SS‐34 rotor (or equivalent; unit 5.1)
  • 1.5‐ml polypropylene microcentrifuge tubes
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Figures

Videos

Literature Cited

Literature Cited
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