Introduction of Plasmid DNA into Cells

Christine E. Seidman1, Kevin Struhl1

1 Harvard Medical School, Boston
Publication Name:  Current Protocols in Protein Science
Unit Number:  Appendix 4D
DOI:  10.1002/0471140864.psa04ds13
Online Posting Date:  May, 2001
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Abstract

This appendix presents a procedure for transformation using calcium chloride, and also an alternate procedure for one‐step preparation and transformation of competent cells.

     
 
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Table of Contents

  • Basic Protocol 1: transformation Using Calcium Chloride
  • Alternate Protocol 1: One‐Step Preparation and Transformation of Competent Cells
  • Literature Cited
     
 
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Materials

Basic Protocol 1: transformation Using Calcium Chloride

  Materials
  • Single colony of E. coli cells
  • LB medium ( appendix 4A)
  • CaCl 2 solution, ice‐cold
  • LB plates containing ampicillin ( appendix 4A)
  • Plasmid DNA ( appendix 4C)
  • Beckman JS‐5.2 rotor or equivalent
NOTE: All materials and reagents coming into contact with bacteria must be sterile.

Alternate Protocol 1: One‐Step Preparation and Transformation of Competent Cells

  • 2× transformation and storage solution (TSS), ice‐cold: dilute sterile 40% (w/v) polyethylene glycol (PEG) 3350 to 20% in sterile LB medium ( appendix 4A) containing 100 mM MgCl 2. Add DMSO to 10% and adjust to pH 6.5.
  • LB medium ( appendix 4A) containing 20 mM glucose
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Figures

Videos

Literature Cited

   Dower, W.J., Miller, J.F., and Ragdale, C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Rs. 16:6127‐6145.
   Hanahan, D. 1983. Studies on transformation of Escherichi coli with plasmids. J. Mol. Biol. 166:557‐580.
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