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Standard Ligation
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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
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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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