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
