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Standard Ligation

互联网

1496
 
 
 
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
 

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
 

incubate for 2-3 h (or ovn) at 14℃.

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:

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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