Preparation of Rice Plant Genomic DNA for Various Applications

Lian Zhou1, Rongxin Shen1, Xingliang Ma1, Heying Li1, Gousi Li1, Yao‐Guang Liu2

1 These authors contributed equally to this work, 2 College of Life Sciences, South China Agricultural University, Guangzhou
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20002
Online Posting Date:  May, 2016
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Abstract

Research on plant molecular biology, genetic engineering, and crop molecular breeding involves various manipulations of plant genomic DNAs (gDNAs). These studies require preparing gDNAs from plant materials using suitable methods according to the yield, intactness, and purity of the resultant gDNA samples. Here we describe protocols for preparation of rice plant gDNAs for various applications including: (1) maxipreps and (2) minipreps of gDNAs for restriction analysis, gene cloning, PCR amplification, genomic sequencing, and genotyping; (3) preparation of megabase‐sized nuclear DNA for construction of large‐insert genomic libraries and long‐range physical mapping; and (4) 96‐well‐format high‐throughput gDNA preparation for PCR‐based genotyping. The methods are also suitable for other plant species including dicots. © 2016 by John Wiley & Sons, Inc.

Keywords: DNA preparation; genomic DNA; nuclear DNA; plants

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Plant gDNA Using CTAB
  • Basic Protocol 2: Mini Preparation of Plant gDNA
  • Basic Protocol 3: Preparation of Megabase Plant gDNA
  • Basic Protocol 4: High‐Throughput Preparation of Plant gDNA for PCR‐Based Genotyping
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Plant gDNA Using CTAB

  Materials
  • Rice plant tissue
  • Liquid nitrogen
  • CTAB extraction buffer (see recipe)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 10% (w/v) CTAB
  • CTAB precipitation buffer (see recipe)
  • High‐salt TE buffer, pH 8.0 (see recipe)
  • 10 mg/ml RNase A (store at −20°C)
  • Isopropanol (or ethanol)
  • 70% ethanol
  • TE buffer, pH 8.0 (no NaCl; see recipe)
  • Lambda DNA
  • Restriction enzyme (e.g., HindIII, EcoRI)
  • Mortars and pestles
  • 50‐ml centrifuge tubes and tube rack
  • 55° and 65°C water baths
  • Shaker
  • Centrifuge (Eppendorf 5810R) and angle rotor (F‐34‐6‐38, with adapters) for 50‐ml centrifuge tubes
  • Spectrophotometer (NanoDrop2000, Thermo Scientific)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 2: Mini Preparation of Plant gDNA

  Materials
  • Rice leaf tissue
  • Tungsten carbide beads (diameter 4 mm or 3 mm)
  • Liquid nitrogen
  • Extraction buffer 1 (see recipe; containing SDS) or extraction buffer 2 (see recipe; containing CTAB)
  • Protein‐denaturation mixture (see recipe)
  • 3 M sodium acetate, pH 5.2
  • Isopropanol
  • 70% ethanol
  • TE buffer, pH 8.0
  • 10 mg/ml RNase A (store at −20°C)
  • 1.5‐ml and 2‐ml microcentrifuge tubes and tube rack
  • FastPrep‐24 Grinder (MP, Biochemicals) or CK1000D Grinder (Thmorgan)
  • Mortars and pestles
  • Phillips screwdriver
  • 65°C water bath
  • Shaker
  • Microcentrifuge
  • Additional reagents and equipment for checking DNA concentration and quality ( protocol 1, steps 11 to 13)

Basic Protocol 3: Preparation of Megabase Plant gDNA

  Materials
  • Seeds of interest
  • Liquid nitrogen
  • NIB/Triton X‐100 (see recipe)
  • Nuclei isolation buffer (NIB; see recipe)
  • 1.8% (w/v) low‐melting agarose prepared in 45 mM Tris borate/1 mM disodium EDTA (pH 8.0)
  • Lysis buffer (see recipe)
  • Phenylmethanesulfonyl fluoride (PMSF)
  • TEN buffer (see recipe)
  • 28° to 30°C growth chamber for seeds
  • Mortar and pestle
  • 500‐ml glass beakers
  • Metal medicinal spoon
  • Nylon meshes with pore sizes of 149 μm, 74 μm, and 37 μm
  • 50‐ml centrifuge tubes
  • Centrifuge (Eppendorf 5810R) and an angle rotor (F‐34‐6‐38) with adapters for 50‐ml centrifuge tubes
  • 45° and 50°C water baths
  • Mold for agarose gel plugs (BioRad or home‐made)
  • Shaker
  • CHEF Mapper AX apparatus (BioRad)
  • Additional reagents and equipment for pulsed‐field gel electrophoresis (Gemmill et al., ) and agarose gel electrophoresis (Voytas, )

Basic Protocol 4: High‐Throughput Preparation of Plant gDNA for PCR‐Based Genotyping

  Materials
  • Seeds of interest
  • Soil
  • Tungsten carbide alloy beads (diameter of 4 mm or 3 mm) with high specific gravity
  • 10× preparation buffer (see recipe)
  • Taq DNA polymerase and buffer
  • dATP, dCTP, dGTP, and TTP
  • Appropriate forward and reverse primers
  • 96‐well deep‐well plates (2.2‐ml well size) and silicone rubber covers
  • 8‐ or 12‐channel pipettor
  • Centrifuge (Eppendorf 5810R) and basket rotor for 96‐well plates
  • 96‐pin replicator (a commercial product, or a home‐made one using #304 stainless steel wire of 1.2‐mm diameter)
  • Additional reagents and equipment for PCR (Kramer and Coen, ), polyacrylamide gel electrophoresis (PAGE; Gallagher, ), and agarose gel electrophoresis (Voytas, )
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Figures

Videos

Literature Cited

Literature Cited
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  Dellaporta, S.L., Wood, J., and Hicks, J.B. 1983. A plant DNA minipreparation: Version 2. Plant Mol. Biol. Rep. 1:19‐22. doi: 10.1007/BF02712670.
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  Wang, H., Chu, Z., Ma, X., Li, R., and Liu, Y.G. 2013. A high through‐put protocol of plant genomic DNA preparation for PCR. Acta Agron. Sin. 39:1200‐1205. doi: 10.3724/SP.J.1006.2013.01200.
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