Team:PKU Beijing/Notebook/Protocol/Ligation

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Notebook > Protocol > Ligation of Inserting DNA into Plasmid Vector DNA

Protocol for ligation of inserting 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/┬Ál
  • ddwater

Procedure:
1. Test the concentration of the DNA sample(s).
2. Pipet the following into a microfuge tube:

Linearized vector DNAaround 100ng
Insert DNA (at 3:1 molar excess over vector)variable
10x ligation buffer1uL
T4 DNA Ligase1uL
ddwaterRest of volume
Total volume10 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.

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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