| - 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 |  |
- 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°C
- 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 | | |  | |