Preparing Lambda DNA from Phage Lysates

Karen Lech1, K.J. Reddy2, L.A. Sherman2

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 2 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.13
DOI:  10.1002/0471142727.mb0113s10
Online Posting Date:  May, 2001
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Abstract

DNA extracted from lambda‐derived vectors is typically subcloned into plasmid or filamentous phage vectors. The first two protocols describe methods for isolating phage DNA from large‐ and medium‐scale liquid lysates. These two methods use either density‐gradient centrifugation or ion‐exchange chromatography to purify the phage particles. The third protocol describes a rapid procedure for isolating phage DNA, suitable for small‐scale liquid lysates.

     
 
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Table of Contents

  • Basic Protocol 1: Preparing DNA by Step‐ and Equilibrium‐Gradient Centrifugation
  • Alternate Protocol 1: Preparing DNA Using DEAE‐Cellulose Column Chromatography
  • Alternate Protocol 2: Preparing DNA from Small‐Scale Liquid Lysates
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing DNA by Step‐ and Equilibrium‐Gradient Centrifugation

  Materials
  • recipe5× polyethylene glycol (PEG) solution
  • Suspension medium (SM; unit 1.11)
  • Potassium chloride
  • recipeCsCl solutions
  • recipeLow‐salt buffer
  • Buffered phenol (unit 2.1)
  • Chloroform
  • 2 M Tris⋅Cl (pH 8.5)/ 0.2 M EDTA (optional, for formamide extraction)
  • Formamide (very high grade, preferably recrystallized; optional)
  • TE buffer, pH 8.0 ( appendix 22)
  • Beckman JA‐10, JA‐20, SW‐28, and VTi50 rotors and bottles/tubes (or equivalents)
  • 3‐ml syringe with 25‐G needle
  • Beckman VTi50 quick‐seal tubes
  • Additional materials for preparing liquid phage lysate (unit 1.12), titering lambda phage (unit 1.11), and quantitation of DNA ( appendix 3D)

Alternate Protocol 1: Preparing DNA Using DEAE‐Cellulose Column Chromatography

  Materials
  • recipeTM buffer
  • 2% sodium dodecyl sulfate (SDS; optional)
  • 0.1 M EDTA (optional)
  • Sodium chloride
  • Polyethylene glycol (PEG) 8000
  • DEAE‐cellulose (microgranular anion exchanger; Whatman DE52 #4057‐050)
  • 0.05 N HCl
  • 10 M NaOH
  • Sodium azide
  • 5 M NaCl
  • Ice‐cold 100% isopropanol
  • TE buffer, pH 8.0 ( appendix 22)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 3 M sodium acetate, pH 6.0
  • 70% ethanol and ice‐cold 100% ethanol
  • Beckman JA‐14 and JA‐20 rotors (or equivalents)
  • 15‐ and 30‐ml Corex centrifuge tubes
  • 10‐ml disposable syringe (1.4 cm‐i.d., optional; Becton Dickinson) or 1.5 × 10–cm standard glass or disposable column ( Bio‐Rad)
  • Glass‐fiber filter or glass wool (optional)
  • Additional reagents and equipment for preparing liquid or plate lysate (unit 1.12), titering lambda phage (unit 1.11), agarose gel electrophoresis (unit 2.5), and phenol extraction/ethanol precipitation (unit 2.1)

Alternate Protocol 2: Preparing DNA from Small‐Scale Liquid Lysates

  Additional Materials
  • 5 mg/ml DNase (unit 3.12)
  • 10 mg/ml DNase‐free RNase (unit 3.13)
  • 0.05 M Tris⋅Cl, pH 8.0
  • 3 M sodium acetate, to pH 4.8 with acetic acid
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Figures

Videos

Literature Cited

Literature Cited
   Benson, S. and Taylor, R.K. 1984. A rapid small‐scale procedure for isolation of phage lambda DNA. Biotechniques 2:126‐127.
   Creaser, E.H. and Taussig, A. 1957. The purification and chromatography of bacteriophages on anion‐exchange cellulose. Virology 4:200‐208.
   Helms, C., Graham, M.Y., Dutchik, J.E., and Olson, M.V. 1985. A new method for purifying lambda DNA from phage lysates. DNA 4:39‐49.
   Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Reddy, K.J., Kuwabara, T., and Sherman, L.A. 1988. A simple and efficient procedure for the isolation of high‐quality phage λ DNA using a DEAE‐cellulose column. Anal. Biochem. 168:324‐331.
   Shuang‐Young, X. 1986. A rapid method for preparing phage λ DNA from agar plate lysates. Gene Anal. Techn. 3:90‐91.
   Webb, R., Reddy, K.J., and Sherman, L.A. 1989. Lambda ZAP: Improved strategies for expression library construction and use. DNA 8:69‐73.
   White, B.A. and Rosenzweig, S. 1989. A reliable method for the purification of bacteriophage λ DNA. Biotechniques 7:694‐695.
Key References
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