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Quick Shotgun Cloning of Phage Inserts

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717

 

  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 NH4 Ac 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
    H2 O 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

 

Solutions:

 

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
H2 O 343.0 ml
Total 1 ml
   
     

 

Remarks:

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.

 

Materials:

Reagent/Tool Supplier Cat.-#
BSA    
ATP    
T4 DNA Ligase    

 

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