Preparation of Plasmid DNA

JoAnne Engebrecht1, Roger Brent2

1 State University of New York, Stony Brook, 2 The Molecular Scienace Institute, Berkeley
Publication Name:  Current Protocols in Protein Science
Unit Number:  Appendix 4C
DOI:  10.1002/0471140864.psa04cs13
Online Posting Date:  May, 2001
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Abstract

This appendix presents basic procedures for alkaline lysis minipreps, both in tubes and in microtiter plates, and also provides an for boiling lysis. A describes storage of plasmid DNA.

     
 
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Table of Contents

  • Basic Protocol 1: Alkaline Lysis Miniprep
  • Alternate Protocol 1: Alkaline Lysis in 96‐Well Microtiter Plates
  • Basic Protocol 2: Boiling Miniprep
  • Support Protocol 1: Storage of Plasmid DNA
     
 
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Materials

Basic Protocol 1: Alkaline Lysis Miniprep

  Materials
  • LB medium or enriched medium(e.g., superbroth or terrific broth; appendix 4A)containing appropriate antibiotic
  • Glucose/Tris/EDTA (GTE) solution: 50 mM glucose/ 25 mM Tris⋅Cl (pH 8.0; appendix 2E)/ 10 mM EDTA ( appendix 2E)
  • TE buffer ( appendix 2E)
  • NaOH/SDS solution: 0.2 M NaOH/1% (w/v) SDS
  • Potassium acetate solution, pH 4.8: 29.5 ml glacial acetic acid/KOH pellets to pH 4.8 (several)/H 2O to 100 ml
  • 95% and 70% ethanol

Alternate Protocol 1: Alkaline Lysis in 96‐Well Microtiter Plates

  • TYGPN medium ( appendix 4A)
  • Isopropanol
  • 96‐well microtiter plates (Dynatech PS plates or equivalent)
  • Multichannel pipetting device (8‐prong Costar; 12‐prong Titer Tek)
  • Multitube vortexer
  • Sorvall RT‐6000 low‐speed centrifuge, or equivalent, with microplate carrier in H‐1000B rotor

Basic Protocol 2: Boiling Miniprep

  Materials
  • LB medium ( appendix 4A) containing appropriate antibiotic
  • STET solution: 8% (w/v) sucrose/0.5% (v/v) Triton X‐100/ 50 mM EDTA ( appendix 2E)/ 50 mM Tris⋅Cl, pH 8.0 ( appendix 2E)
  • Hen egg white lysozyme
  • Isopropanol, ice‐cold
  • TE buffer ( appendix 2E)
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Literature Cited

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