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Standard Ligation

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Standard Ligation
Reference:   Schneitz Lab Last updated: 1/27/00 By: Kay Schneitz
     
 
  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    

 

 
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