Genomic DNA Isolation from Maize (Zea mays) Leaves Using a Simple, High‐Throughput Protocol

Kristen A. Leach1, Paula C. McSteen2, David M. Braun1

1 Division of Biological Sciences, Interdisciplinary Plant Group, Missouri Maize Center, University of Missouri, Columbia, Missouri, 2 Bond Life Sciences Center, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20000
Online Posting Date:  May, 2016
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Abstract

A simple, robust, inexpensive, high‐throughput method for isolating genomic DNA from maize (Zea mays) leaf tissues is described. The DNA obtained using this extraction protocol is suitable for polymerase chain reaction (PCR) genotyping, which can be employed for the identification of alleles in diverse genetic and breeding approaches, such as marker‐assisted selection, genetic fine mapping, and mutant introgression. This method utilizes 96‐well plates for the collection of leaf tissue and the subsequent isolation of genomic DNA. The DNA isolation step is performed inexpensively within 3 hr and uses a urea‐based extraction buffer that does not require an organic extraction step. Yields of genomic DNA are sufficient to perform ∼25 PCR‐genotyping reactions per sample. These qualities, coupled with the protocol being robust and easy for inexperienced users to master, make this method ideal for new researchers. © 2016 by John Wiley & Sons, Inc.

Keywords: genomic DNA; high‐throughput; maize leaf; urea‐based extraction

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Maize leaf tissue (freshly harvested or frozen at −80°C)
  • Urea extraction buffer (see recipe)
  • 1:10 ammonium acetate:isopropanol solution (see recipe)
  • 70% ethanol
  • Deionized water suitable for PCR
  • 10 mM Tris·Cl, pH 8.0 (optional; for long‐term storage)
  • 3‐mm stainless steel balls (Union Process, cat. no. 0070‐01, 1/8 in.)
  • 1.1‐ml MicroTubes in a MicroPlate (National Scientific Supply, cat. nos. TN0946‐12R, TN0946‐12B)
  • MicroCaps (National Scientific Supply, cat. no. TN0946‐12 C)
  • 6.35‐mm hole punch
  • Fine point forceps
  • 300 μl multichannel pipettor
  • 300 μl barrier pipet tips (MidSci, cat. no. AV300)
  • Reagent reservoir
  • Mini BeadBeater (BioSpec Products, cat. no. 1001)
  • Centrifuge with adaptor for 96‐well plates
  • 1‐ml polypropylene deep well plates (Fisher Scientific, cat. no. 12‐566‐120)
  • AxyMat Microplate Sealing Mat (Axygen Scientific, cat. no. AM‐2ML‐RD)
  • Vortex
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Figures

Videos

Literature Cited

Literature Cited
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