Introduction of Plasmid DNA into Cells

Christine E. Seidman1, Kevin Struhl1, Jen Sheen2, Timm Jessen3

1 Havard Medical School, Boston, Massachusetts, 2 Massachusetts General Hospital, Boston, Massachusetts, 3 Hoechst AG, Frankfurt am Main, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.8
DOI:  10.1002/0471142727.mb0108s37
Online Posting Date:  May, 2001
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Abstract

Transformation of E. coli can be achieved using any of the four protocols in this unit. The first method using calcium chloride gives good transformation efficiencies, is simple to complete, requires no special equipment, and allows storage of competent cells. The alternate oneā€step method is considerably faster and also gives good transformation efficiencies (although they are somewhat lower). If considerably higher transformation efficiencies are needed, the third method using electroporation is simple, fast, and reliable. As in the calcium chloride protocol, prepared cells can be stored. The final method described is an adaptation of the electroporation protocol that allows direct transfer of vector DNA from yeast into E. coli.

     
 
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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
  • Basic Protocol 2: High‐Efficiency Transformation by Electroporation
  • Alternate Protocol 2: Direct Electroporetic Transfer of Plasmid DNA from Yeast into E. Coli
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Transformation Using Calcium Chloride

  Materials
  • Single colony of E. coli cells
  • LB medium (unit 1.1)
  • recipeCaCl 2 solution (see recipe), ice cold
  • LB plates (unit 1.1) containing ampicillin (Table 97.80.4711)
  • Plasmid DNA (units 1.6 & 1.7)
  • Chilled 50‐ml polypropylene tubes
  • Beckman JS‐5.2 rotor or equivalent
  • 42°C water bath
  • Additional reagents and equipment for growth of bacteria in liquid media (unit 1.2)
NOTE: All materials and reagents coming into contact with bacteria must be sterile.

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

  • recipe2× transformation and storage solution (TSS; see recipe), ice cold
  • recipeLB medium (unit 1.1) containing 20 mM glucose

Basic Protocol 2: High‐Efficiency Transformation by Electroporation

  Materials
  • Single colony of E. coli cells
  • LB medium (unit 1.1)
  • H 2O, ice cold
  • 10% glycerol, ice cold
  • recipeSOC medium (see recipe)
  • LB plates (unit 1.1) containing antibiotics (Table 97.80.4711)
  • 1‐liter centrifuge bottle, 50‐ml narrow‐bottom polypropylene tube, and microcentrifuge tubes, chilled ice cold
  • Beckman J‐6M centrifuge (or equivalent)
  • Beckman JS‐4.2 rotor (or equivalent) and adaptors for 50‐ml narrow‐bottom tubes
  • Electroporation apparatus with a pulse controller or 200‐ or 400‐ohm resistor
  • Chilled electroporation cuvettes, 0.2‐cm electrode gap
  • Additional reagents and equipment for growth of bacteria in liquid media (unit 1.2)
NOTE: All materials and reagents coming into contact with bacteria must be sterile.

Alternate Protocol 2: Direct Electroporetic Transfer of Plasmid DNA from Yeast into E. Coli

  • Single colony of E. coli KC8 cells (unit 20.1)
  • Streak colony of Trp− plasmid–harboring EGY48 yeast cells on Gal/Raff/Xgal/CM plates (unit 20.1), no older than 2 weeks
  • M9 minimal medium and plates (unit 1.1) containing 100 µg/ml ampicillin (Table 97.80.4711) and standard concentrations of leucine, histidine, and uracil
  • Additional reagents and equipment for growth and manipulation of yeast (unit 13.2) and for plasmid DNA miniprep (unit 1.6) or PCR (unit 15.1)
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Figures

Videos

Literature Cited

Literature Cited
   Calvin, N.M. and Hanawalt, P.C. 1988. High‐ efficiency transformation of bacterial cells by electroporation. J. Bacteriol. 170:2796‐2801.
   Chung, C.T., Niemela, S.L., and Miller, R.H. 1989. One‐step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U.S.A. 86:2172‐2175.
   Dagert, M. and Ehrlich, S.D. 1974. Prolonged incubation in calcium chloride improves competence of Escherichia coli cells. Gene 6:23‐28.
   Dower, W.J., Miller, J.F., and Ragsdale, C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res. 16:6127‐6145.
   Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557‐580.
   Kushner, S.R. 1978. An improved method for transformation of Escherichia coli with Col EI derived plasmids. In Genetic Engineering (H.W. Boyer and S. Nicosia, eds.) pp. 17‐ 23. Elsevier/North Holland, Amsterdam.
   Mandel, M. and Higa, A. 1970. Calcium‐dependent bacteriophage DNA infection. J. Mol. Biol. 53:159‐162.
   Marcil, R. and Higgins, D.R. 1992. Direct transfer of plasmid DNA from yeast to E. coli by electroporation. Nucl. Acids Res. 20:917.
   Miller, J.F., Dower, W.J., and Tompkins, L.S. 1988. High‐ voltage electroporation of bacteria: Genetic transformation of Campylobacter jejuni with plasmid DNA. Proc. Natl. Acad. Sci. U.S.A. 85:856‐860.
   Shigekawa, K. and Dower, W.J. 1988. Electroporation of eukaryotes and prokaryotes: A general approach to the introduction of macromolecules into cells. BioTechniques 6:742‐751.
Key References
   Dower et al., 1988. See above.
  The paper from which the second basic protocol was derived, and the highest‐efficiency E. coli transformation by electroporation published to date.
   Hanahan, 1983. See above.
  An extremely thorough explanation of the parameters affecting transformation efficiency.
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