Team:PKU Beijing/Notebook/Protocol/Ligation

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

Protocol for ligation of inserting DNA into plasmid vector DNA
 [[Media:PKU_Protocol_for_ligation_of_insert_DNA_into_plasmid_vector_DNA.pdf|download PDF version]]

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: 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.