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

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 H2O 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