From 2009.igem.org
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Revision as of 08:09, 7 October 2009
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Protocol for ligation of insert DNA into plasmid vector DNA
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Materials:
- DNA sample(s) in water or TE buffer
- 10x ligation buffer
- T4 DNA Ligase, 5 u/µl
- ddwater
Procedure:
1. Test the concentration of the DNA sample(s).
2. Pipet the following into a microfuge tube:
| Linearized vector DNA | around 100ng
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| Insert DNA (at 3:1 molar excess over vector) | variable
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| 10x ligation buffer | 1uL
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| T4 DNA Ligase | 1uL
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| ddwater | Rest of volume
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| Total volume | 10 uL
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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.
Tips:
- 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.
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