Standard Ligation
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Prepare a ligation mix:
| Ligation Mix (2x) | |
| 10x ligase buffer | 1.0 ml |
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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 | ||
