Quick Shotgun Cloning of Phage Inserts

 

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