Preparing and Using M13‐Derived Vectors

David Greenstein1, Claude Besmond2

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 2 Centre de Recherche Inserm, Paris, France
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.15
DOI:  10.1002/0471142727.mb0115s09
Online Posting Date:  May, 2001
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Abstract

Many vectors in current use are derived from filamentous phages. These vectors are used because DNA inserted into them can be recovered in two forms: double‐stranded circles and single‐stranded circles. This overview unit describes the lifecycle of filamentous phages along with the development and use of filamentous phage vectors.

     
 
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Table of Contents

  • Basic Protocol 1: Isolating Single M13‐Derived Vectors
  • Basic Protocol 2: Preparing Single‐Stranded Phage DNA from M13‐Derived Vectors
  • Basic Protocol 3: Preparing Double‐Stranded Replicative‐Form DNA
  • Basic Protocol 4: Preparing Single‐Stranded DNA from Plasmids Using Helper Phage
  • Basic Protocol 5: Introduction of Phage DNA into Cells
  • Basic Protocol 6: Determining Size of Inserts in Single‐Stranded Vectors
  • Support Protocol 1: Determining Insert Orientation
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Isolating Single M13‐Derived Vectors

  Materials
  • E. coli strain infected with M13‐derived vector (e.g., JM101 infected with M13mp18)
  • 2× TY medium (unit 1.1)
  • 20 mg/ml IPTG in H 2O (stored in aliquots at −20°C)
  • 20 mg/ml Xgal in dimethylformamide (stored in aliquots at −20°C)
  • 45°C top agarose (unit 1.1)
  • H plates, prewarmed to 37°C (unit 1.1)
  • 5‐ml Falcon tubes with caps or equivalent plastic tubes
  • 37°C incubator
NOTE: All materials coming into contact with E. coli must be sterile.

Basic Protocol 2: Preparing Single‐Stranded Phage DNA from M13‐Derived Vectors

  Materials
  • E. coli strain (male‐type, e.g., JM105)
  • M13‐derived vector
  • 2× TY medium (unit 1.1)
  • PEG solution (unit 1.7)
  • TE buffer ( appendix 22)
  • Buffered phenol (unit 2.1)
  • 3 M sodium acetate
  • 100% ethanol and cold 70% ethanol
  • Sterile toothpicks
  • 37°C shaking water bath
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)

Basic Protocol 3: Preparing Double‐Stranded Replicative‐Form DNA

  Materials
  • F+ or Hfr E. coli strain
  • 2× TY medium (unit 1.1)
  • Recombinant phage
  • 20% glucose
  • 1 mg/ml chloramphenicol in ethanol, freshly prepared
  • Sorvall SS‐34 rotor or equivalent

Basic Protocol 4: Preparing Single‐Stranded DNA from Plasmids Using Helper Phage

  Materials
  • F+ or Hfr E. coli strain (Table 97.80.4711) containing a plasmid (pUC118, pBS, or equivalent; see commentary)
  • 2× TY medium (unit 1.1)
  • 37°C shaking water bath and 65°C water bath
  • Sorvall SS‐34 rotor or equivalent

Basic Protocol 5: Introduction of Phage DNA into Cells

  Materials
  • 2% sodium dodecyl sulfate (SDS)
  • recipeLoading buffer

Basic Protocol 6: Determining Size of Inserts in Single‐Stranded Vectors

  Additional Materials
  • 5 M NaCl
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Figures

Videos

Literature Cited

Literature Cited
   Enea, V. and Zinder, N.D. 1982. Interference resistant mutants of phage f1. Virology. 122:222‐226.
   Howarth, A.J., Gardner, R.C., Messing, J., and Sheperd, R.J. 1981. Nucleotide sequence of a naturally occurring deletion of a mutant of a cauliflower mosaic virus. Virology. 112:678.
   Messing, J. 1983. New M13 vectors for cloning. Methods Enzymol. 101:20‐78.
   Messing, J., Gronenborn, B., Muller‐Hill, B., and Hofschneider, P.H. 1977. Single‐strand filamentous DNA phage as a carrier for in vitro recombined DNA. Proc. Natl. Acad. Sci. U.S.A. 74:3642‐3646.
   Russell, M., Kidd, S., and Kelley, M.R. 1986. An improved filamentous helper phage for sequencing single‐stranded plasmid DNA. Gene. 45:333‐338.
   Viera, J. and Messing, J. 1987. Production of single‐stranded plasmid DNA. Methods Enzymol. 153:3‐11.
Key References
   Dente, L., Sollazzo, M., Cesareni, G., and Cortese, R. 1985. The pEMBL family of single‐stranded vectors. In DNA cloning: A Practical Approach (D.M. Glover, ed.) pp. 101‐108. IRL Press, Oxford.
  Reviews biology of non‐M13−derived single‐stranded vectors.
   Howarth, et al 1981. See above
  Describes techniques for detrmining insert orientation.
   Messing, J. 1983. See above
  Maintenance, propagation, and titration of filamentous phage vectors are extensively described.
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