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Quick Shotgun Cloning of Phage Inserts

Zürich Online Bibliothek Uni ZH Vorlesungen Institute of Plant Biology Home Page U of Zürich Home Page   Schneitz Lab Home Page / Methods / Molecular Cloning / Quick Shotgun Cloning of Phage Inserts
Quick Shotgun Cloning of Phage Inserts
Reference:  Schneitz Lab Last updated: 1/27/00 By: Kay Schneitz
  1. digest genomic phage DNA:

    EcoRI Digestion
    10x EcoRI buffer 0.5 ml
    phage DNA (200 ng) 4.5 ml
    Eco RI (20 u/ml) 0.2 ml
    total 5.2 ml

  2. incubate for 15 min at 37°C (heating block or water bath)
  3. heat for 5 min at 65°C (heating block)
  4. add 2 ml 5 M NH4Ac and 10 ml isopropanol
  5. spin for 20 min at 4°C
  6. wash pellet with 70% EtOH (-20°C), speed vac and dissolve pellet in 4 ml 0.1 x TE (ca 50 ng/ml)
  7. prepare a ligation mix:

    Ligation Mix (2x)
    10x ligase buffer 1.0 ml
    digested vector
    (0.1 mg/ml)
    1.0 ml
    H2O 6.0 ml
    total 8.0 ml

  8. divide ligation mix between two Eppendorf tubes

    Ligation Rxn
      Insert Control
    ligation mix 4.0 ml 4.0 ml
    insert 1.0 ml --- ml
    T4 DNA ligase (400 u/ml) 0.2 ml 0.2 ml
    Total 5.2 ml 4.2 ml

  9. incubate for 2-3 h (or ovn) at 14°C
  10. proceed with the transformation of the appropriate E. coli strain




10x Ligation Buffer:

0.5 M Tris-HCl pH 7.8, 50 mM MgCl2, 0.1 M b-mercaptoethanol, 50 mM ATP, 5 mg/ml BSA

Ligation Buffer (10x)
1 M Tris-HCl pH 7.8 500.0 ml
1 M MgCl2 50.0 ml
b-mercaptoethanol 7.0 ml
100 mM ATP 50.0 ml
0.1 g/ml BSA 50.0 ml
H2O 343.0 ml
Total 1 ml



Quickprep phage DNA gets prepared according to our standard protocol. We use Lambda DASH or EMBL4 lambda phages as vectors. Thus the insert gets released using EcoRI. Be aware that the insert usually carries internal EcoRI sites as well.



Reagent/Tool Supplier Cat.-#
T4 DNA Ligase    



  © Copyright 1997-2000, Kay Schneitz. Last updated 3/24/99; 1:10:39 PM. Send comments to the webmeister.