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Protocol for the quick ligation with T4 DNA ligase
Reaction conditions:
66 mM Tris-HCl, pH 7.6
6.6 mM MgCl2
10 mM DTT
1 mM ATP
8% PEG 6000
1-25 U/ul T4 DNA ligase
1. Combine 25-100 ng of the vector with a 3-fold molar excess of the insert
2. Adjust the volume to 11 ul with H2O
3. Add 2ul of 10x ligation buffer (Takara)
2 ul 10 mM ATP
4 ul 40% PEG 6000 and mix
4. Add 1 ul of T4 DNA ligase (Takara) and mix thoroughly.
5. Centrifuge briefly and incubate at room temperature for 10-15 min
6. Chill on ice, then transform 1-10ul of the ligated products into competent cells or store at -20oC
Controls (optional):
1. Set up and analyze parallel ligation reaction lacking an insert component.
In a successful hybrid construction, the number of colonies obtained when all DNA components are present should be higher than that of the control reaction. As a general rule, 1 ng of ligated DNA should generate from 10 to 100 transformants
2. Incubate an aliquot of competent cells in the absence of DNA. The appearance of transformants is indicative of a contamination in the cells or transformation buffers.
3. You may want to measure the transformation efficiency by incubating another aliquot of competent cells with 1 ng of an intact plasmid DNA. 1000 colonies should be achieved easily.
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