Generation of Transformation Competent E. coli

Nicholas Renzette1

1 Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3L
DOI:  10.1002/9780471729259.mca03ls22
Online Posting Date:  August, 2011
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Abstract

Transformation of bacterial cells, specifically Escherichia coli, is a widespread and powerful laboratory technique. This unit describes two protocols to make competent E. coli, which can then be used in subsequent transformations. The first protocol describes the preparation of chemically competent E. coli. These cells can be transformed in almost any laboratory with simple equipment, such as a water bath. The second protocol is used to make electrocompetent cells. These cells will yield high numbers of transformants, but require an electroporator, which may not be available in all environments. Thus, these two protocols should be sufficient for almost any situation that requires the preparation of competent E. coli. Curr. Protoc. Microbiol. 22:A.3L.1‐A.3L.5. © 2011 by John Wiley & Sons, Inc.

Keywords: competent cells; E. coli; transformation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Chemically Competent Cells
  • Basic Protocol 2: Preparation of Electrocompetent Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Chemically Competent Cells

  Materials
  • Single E. coli colony
  • Luria‐Bertani (LB) broth (see recipe)
  • Transformation buffer I (TB‐I), 4°C (see recipe)
  • Transformation buffer II (TB‐II), 4°C (see recipe)
  • 37°C incubator with shaker
  • 50‐ml polypropylene centrifuge tubes
  • Ice‐water bath (4°C)
  • Sorvall RC‐5B Superspeed centrifuge or similar
  • Sorvall SS‐34 rotor or similar
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Preparation of Electrocompetent Cells

  Materials
  • Single E. coli colony
  • LB broth
  • 10% (v/v) glycerol, 4°C (see recipe)
  • 37°C incubator with shaker
  • Sorvall RC‐5B superspeed centrifuge or similar
  • Sorvall GSA rotor or similar
  • 250‐ml polypropylene centrifuge tubes or similar
  • 1.5‐ml microcentrifuge tubes
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Figures

Videos

Literature Cited

Literature Cited
   Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 2001. Introduction of Plasmid DNA into Cells. John Wiley & Sons, Inc., New York.
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