Construction of Bacteriophage P1 Libraries with Large Inserts

Nat Sternberg1, Nancy S. Shepherd2

1 Du Pont/Merck Pharmaceuticals, Glenolden, Pennsylvania, 2 Glaxo Wellcome, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.3
DOI:  10.1002/0471142905.hg0503s09
Online Posting Date:  May, 2001
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Abstract

The bacteriophage P1 cloning system was originally developed as an alternative to YAC and cosmid systems for cloning high‐molecular‐weight genomic DNA. This unit details the preparation of the bacteriophage P1 library. Three support protocols provide the raw materials for the basic procedure, including the vector (pAd10sacBII), the mammalian DNA inserts, and the two packaging extracts that contain the viral proteins necessary to construct a P1 bacteriophage incorporating the vector and insert. A fourth support protocol describes how to induce replication of the plasmids cloned in the basic protocol, isolate the cloned DNA, and analyze the final products.

     
 
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Table of Contents

  • Basic Protocol 1: Cloning Insert DNAUsing P1 Bacteriophage
  • Support Protocol 1: Alkaline Lysis Preparation and CsCl Purification of Plasmid Vector DNA
  • Support Protocol 2: Preparation of Genomic DNA Fragments for Cloning
  • Support Protocol 3: Preparation of P1 Packaging Extracts
  • Support Protocol 4: Modified Alkaline Lysis Preparation of P1 Recombinant DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cloning Insert DNAUsing P1 Bacteriophage

  Materials
  • pAd10sacBII vector DNA purified by alkaline lysis (see protocol 2)
  • ScaI restriction endonuclease and 10× buffer (New England Biolabs)
  • TE buffer, pH 8.0 ( appendix 2D)
  • Buffered phenol, pH 8.0 ( appendix 3B)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 3 M sodium acetate, pH 5.2 ( appendix 2D)
  • 100% and 75% ethanol
  • 50 mM Tris‐Cl (pH 8.0)/ 0.1 mM EDTA
  • recipe10× stop/loading buffer (see recipe)
  • 50 U/µl bacterial alkaline phosphatase (BAP; Life Technologies)
  • BamHI restriction endonuclease and 1× buffer (New England Biolabs; dilute 10× buffer just before use)
  • Calf intestine alkaline phosphatase (CIP; Du Pont NEN)
  • Size‐selected, Sau3AI‐digested genomic DNA (see protocol 3)
  • 400,000 U/ml T4 DNA ligase (in cohesive‐end units; New England Biolabs) and 10× T4 DNA ligase buffer ( appendix 2D)
  • 100 mM Tris⋅Cl (pH 8.0)/250 mM NaCl/100 mM MgCl 2
  • 1 mM 4dNTP mix (1 mM each dATP, dGTP, dCTP, dTTP; appendix 2D)
  • 30 mM dithiothreitol (DTT)
  • 50 mM ATP, pH 7.5 (Boehringer Mannheim)
  • Stage I P1 packaging extract (See protocol 4)
  • recipe1× stage II packaging buffer (see recipe)
  • Stage II packaging extract (See protocol 4)
  • recipeTMGD buffer (see recipe)
  • LB plate bearing well‐isolated colonies of E. coli NS3529 (Table 5.3.3; see )
  • LB medium ( appendix 2D) with and without 5 mM CaCl 2
  • LB plates ( appendix 2D) containing 25 µg/ml kanamycin
  • LB plates ( appendix 2D) containing 25 µg/ml kanamycin and 5% (w/v) sucrose
  • Sorvall H1000B rotor or equivalent
  • 16° to 18°, 30°, 65°, and 70°C water baths
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA ( appendix 3C), FIGE (see protocol 3), and alkaline lysis preparation of P1 recombinant DNA (see protocol 5)

Support Protocol 1: Alkaline Lysis Preparation and CsCl Purification of Plasmid Vector DNA

  Materials
  • E. coli NS3622 (Table 5.3.1)
  • LB medium ( appendix 2D) with and without 25 µg/ml kanamycin
  • TE buffer, pH 8.0 ( appendix 2D)
  • Calcium‐competent (CPMB UNIT ) E. coli NS3529 (Table 5.3.1)
  • LB plates ( appendix 2D) containing 25 µg/ml kanamycin
  • LB plates ( appendix 2D) containing 25 µg/ml kanamycin and 5% (w/v) sucrose
  • Tris/sucrose solution: 50 mM Tris⋅Cl (pH 8.0)/25% (w/v) sucrose, 4°C
  • recipe10 mg/ml lysozyme solution (see recipe)
  • 0.25 M EDTA, pH 8.0 ( appendix 2A)
  • recipeLysis buffer (see recipe)
  • 5 M NaCl
  • Isopropanol, ice cold
  • recipeNTE buffer (see recipe)
  • Biological‐grade cesium chloride (CsCl, e.g., Gallard‐Schlesinger)
  • 10 mg/ml ethidium bromide ( appendix 2D)
  • CsCl‐saturated isopropanol (prepare by adding solid CsCl to isopropanol until the CsCl no longer dissolves)
  • SpeI restriction endonuclease and 10× buffer (New England Biolabs)
  • 30°C incubator with shaker
  • 350‐ml plastic centrifuge bottles
  • 50‐ml Oak Ridge screw‐cap centrifuge tubes
  • Dry ice/ethanol bath
  • Sorvall RC‐5C centrifuge with GSA rotor or equivalent, 4°C
  • Sorvall SS‐34 rotor or equivalent, 4°C
  • Sorvall RC‐70 ultracentrifuge with T‐1270 rotor or equivalent, 20°C
  • 18‐G needle
  • 6‐ or 15‐ml polypropylene snap‐cap tubes
  • Additional reagents and equipment for alkaline lysis preparation of P1 recombinant DNA (see protocol 5), phenol extraction and ethanol precipitation of DNA ( appendix 3C), dialysis (CPMB APPENDIX ), agarose gel electrophoresis (unit 2.7), and transfection of calcium‐competent E. coli (CPMB UNIT )

Support Protocol 2: Preparation of Genomic DNA Fragments for Cloning

  Materials
  • Cultured mammalian cells
  • PBS ( appendix 2D)
  • recipeNTE buffer (see recipe)
  • 10% (w/v) SDS
  • 10 mg/ml proteinase K (Boehringer Mannheim)
  • TE buffer, pH 8.0 ( appendix 2D)
  • 0.1 mM phenylmethylsulfonylfluoride (PMSF; Life Technologies and unit 5.1) in TE buffer, pH 8.0 (prepare fresh)
  • Sau3AI restriction endonuclease (New England Biolabs) and magnesium‐free recipe10× Sau3AI buffer (see recipe)
  • 3 mg/ml nuclease‐free bovine serum albumin (BSA; New England Biolabs)
  • 0.1 M MgCl 2
  • 0.2 M EDTA, pH 8.0 ( appendix 2D)
  • recipe10× stop/loading buffer (see recipe)
  • 1% SeaKem GTG agarose gel (FMC Bioproducts) prepared in 0.5× TBE buffer
  • DNA molecular size markers (see Table 5.3.3)
  • 0.5× TBE buffer ( appendix 2D) with and without 20 µg/ml ethidium bromide
  • NaCl‐saturated butanol (prepare by adding solid NaCl to butanol until the NaCl no longer dissolves)
  • 50‐ml conical centrifuge tubes
  • Sorvall H1000B rotor or equivalent, 4°C
  • 17‐ml polyallomer centrifuge tubes (Beckman)
  • Beckman SW‐27.1 rotor or equivalent, 4°C
  • 30°, 60°, and 70°C water baths
  • Dialysis membrane, MWCO 1,000,000 (Spectrum; CPMB APPENDIX 3)
  • 24‐well microtiter plates, 1.5‐ml microtiter tubes, or 6‐ml Falcon tubes
  • 18‐G needle
  • 0.025‐µm pre‐size dialysis membrane discs, large size (Millipore type VS)
  • 35‐mm petri dishes
  • Polypropylene tubes with caps
  • Additional reagents and materials for dialysis (CPMB APPENDIX 3) partial restriction digestion of DNA (unit 5.2), quantitation of DNA ( appendix 3D), (CPMB UNIT ) or CHEF electrophoresis (unit 5.1), and size fractionation of DNA using sucrose gradients (CPMB UNIT )
    Table 5.3.3   MaterialsDNA Molecular Size Markers

    Marker Size or range (kb) Supplier
    DNA monocut mix 1.5–48 New England Biolabs
    DNA digested with HindIII 2–23 New England Biolabs
    Midrange I 15–300 New England Biolabs
    Midrange II 24–300 New England Biolabs
    T5 DNA digested with SacI and XhoI 25, 78, and 102
    T5 DNA 122 Sigma
    T4 DNA 165 Sigma
    Yeast chromosomes in agar plugs 225 c Bio‐Rad

     cThe smallest yeast chromosome is 225 kb.

Support Protocol 3: Preparation of P1 Packaging Extracts

  Materials
  • E. coli NS3208 and NS3690 (Table 5.3.1)
  • LB plates and LB medium ( appendix 2D) containing 25 µg/ml chloramphenicol, 30° to 32°C
  • LB medium ( appendix 2D)
  • recipePacase buffer, 4°C (prepare fresh; see recipe)
  • LB plates and LB medium ( appendix 2D) containing 25 µg/ml chloramphenicol and 20 µg/ml spectinomycin, warmed
  • 50% (w/v) sucrose, 4°C
  • 50 mM Tris⋅Cl (pH 8.0)/10% (w/v) sucrose, 4°C
  • recipe10 mg/ml lysozyme solution, 4°C (prepare fresh; see recipe)
  • Liquid nitrogen
  • 30°, 32°, and 42°C incubators with shakers
  • Sorvall GSA and SS‐34 rotors with 350‐ml plastic centrifuge bottles, 4°C
  • Ice‐water bath
  • Sonicator (Branson)
  • 4‐ml test tubes (Becton Dickinson Labware; Falcon)
  • 0.5‐ml microcentrifuge tubes

Support Protocol 4: Modified Alkaline Lysis Preparation of P1 Recombinant DNA

  Materials
  • LB/kanamycin/sucrose plates bearing well‐isolated P1 colonies (see protocol 1)
  • LB medium ( appendix 2D) containing 25 µg/ml kanamycin
  • 0.1 M IPTG (store at −20°C; optional)
  • recipeTGE buffer, pH 8.0 (see recipe), ice cold
  • recipe10 mg/ml lysozyme solution (see recipe)
  • 0.2 N NaOH/ 1% (w/v) SDS (prepare fresh)
  • recipePotassium acetate solution, pH 4.8 (see recipe), ice cold
  • 100% and 70% ethanol, −20°C
  • TE buffer, pH 8.0 ( appendix 2D)
  • 2 mg/ml RNase I ( appendix 2D) in TE buffer, pH 8.0 (preincubated 5 min at 95°C; optional)
  • BglII orNotI restriction endonuclease and buffer (e.g., New England Biolabs)
  • recipe10× stop/loading buffer (see recipe)
  • 38°C incubator with shaker
  • 70°C water bath
  • Additional reagents and equipment for pulsed‐field gel electrophoresis (see protocol 3), and standard agarose gel electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

Literature Cited
   Abremski, K., Hoess, R., and Sternberg, N. 1983. Studies on the properties of P1 site‐specific recombination: Evidence for topological unlinked products following recombination. Cell 32:1301‐1311.
   Austin, S., Hart, F., Abeles, A., and Sternberg, N. 1982. Genetic and physical map of a P1 miniplasmid. J. Bacteriol. 152:63‐71.
   Coren, J.S., Pierce, J.C., and Sternberg, N. 1995. Headful packaging revisited: The packaging of more than one molecule into a bacteriophage P1 head. J. Mol. Biol. 249:176‐184.
   Eliason, J.L. and Sternberg, N. 1987. Characterization of the binding sites of c1 repressor of bacteriophage P1: Evidence for multiple asymmetric sites. J. Mol. Biol. 198:281‐293.
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