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 |
|
|