DNA Assembly for Plant Biology

Nicola J. Patron1

1 The Earlham Institute, Norwich Research Park, Norwich, Norfolk
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20038
Online Posting Date:  December, 2016
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Parallel DNA assembly methods allow multiple fragments of DNA to be compiled in a desired order in a single reaction. Several methods enable the efficient one‐step assembly of multiple DNA parts into a suitable plasmid acceptor at high efficiency. Type IIS‐mediated assembly offers the specific advantage of a one‐step reaction that does not require proprietary reagents or the amplification and purification of linear DNA fragments. Instead, multiple plasmids housing standardized DNA parts of interest are combined in an enzyme cocktail. To make these standard parts, DNA sequences with defined functions are assigned specific sequence features. This allows parts to be interoperable and reusable. The availability of collections of DNA parts and molecular toolkits that allow the facile assembly of multigene binary constructs and the establishment of standards for the creation of new parts means Type IIS‐mediated assembly has become a powerful technology for modern plant molecular biologists. © 2016 by John Wiley & Sons, Inc.

Keywords: DNA assembly; gene cloning; modular cloning; biological standards

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Assembly of Standard Parts in the Common Syntax into Transcriptional Units
  • Support Protocol 1: Making New Standard Parts
  • Basic Protocol 2: Assembly of Transcriptional Units into Multi‐Gene Constructs Using MoClo
  • Alternate Protocol 1: Assembly of Transcriptional Units into Multigene Constructs Using GoldenBraid
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Assembly of Standard Parts in the Common Syntax into Transcriptional Units

  • Purified DNA of acceptor plasmid in which parts will be assembled
  • Purified plasmid DNA containing DNA parts to be assembled
  • 10× T4 ligase buffer (e.g., New England BioLabs)
  • 200 U/µl T4 DNA ligase (e.g., New England BioLabs)
  • 1 mg/ml bovine serum albumin (BSA; e.g., New England BioLabs)
  • 10 U/µl BsaI (Eco31I; e.g., Thermo Fisher Scientific)
  • Electrocompetent cells (>109 colony forming units [CFU]/µg) or chemically competent cells
  • LB agar plates supplemented with 0.5 mM IPTG, 0.02 mg/ml X‐gal, and appropriate antibiotics (see Elbing and Brent, )
  • LB medium supplemented with appropriate antibiotics (see Elbing and Brent, )
  • Thermal cycler
  • Electroporator (for electrocompetent cells) or 42°C water bath (for chemically competent cells)
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Literature Cited

Literature Cited
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