1. 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

   
   
   
2. divide ligation mix into 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

   
   
   
3. incubate for 2-3 h (or ovn) at 14°C
4. 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:

As usually when pipetting enzyme reactions keep the tubes on ice during all manipulations. Cip-treat a digested vector cut with only a single enzyme. If the vector was double-digested: phenolize prior to ligating. Use equimolar amounts of vector and insert. As a rule we use 50 ng vector (3 kb) and 50 ng insert (3 kb). More values are given in the table below. Recalculate accordingly when using different-length vectors. Don't use less than 8 ng or so for fragments smaller than 0.5 kb. In the case of small fragments circularization occurs and removes the F from the rxn.

F Used in Standard Ligation
vector (50 ng, 3kb)
Length of F (kb)	Amount of F (ng)
0.5	8.3
1	16.7
1.5	25
2	33.3
2.5	41.7
3	50
3.5	58
4	66.7
5	83.3
6	100
7	116.3

Materials:

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