Notebook > Protocol > Ligation of Inserting DNA into Plasmid Vector DNA
Protocol for ligation of inserting DNA into plasmid vector DNA
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- DNA sample(s) in water or TE buffer
- 10x ligation buffer
- T4 DNA Ligase, 5 u/µl
1. Test the concentration of the DNA sample(s).
2. Pipet the following into a microfuge tube:
|Linearized vector DNA||around 100ng
|Insert DNA (at 3:1 molar excess over vector)||variable
|10x ligation buffer||1uL
|T4 DNA Ligase||1uL
|ddwater||Rest of volume
|Total volume||10 uL
3. Vortex and spin briefly to collect drops.
4. Incubate the mixture at 16 degree for 60-120 min.
5. Use the ligation mixture for transformation.
- Thoroughly mix the 10x ligation buffer before use.
- The optimal insert/vector molar ratio is 3:1.
- To minimize recircularization of the cloning vector, dephosphorylate linearized plasmid DNA with Alkaline Phosphatase(CIAP) prior to ligation. Heats inactivate the phosphatase or remove from the mixture after the dephosphorylation step.
- DNA purity is an important factor for successful ligation. Plasmids should be purified using a method that will ensure isolation of high quality DNA. Use only high quality agarose and fresh electrophoresis buffers for gel-purification of DNA fragments.