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   Protocol by PKU 2010


         Notebook > Protocols > Protocol for ligation of insert DNA into plasmid vector DNA

Contents 1 Protocol for Ligation of Insert DNA into Plasmid Vector DNA 1.1 Materials: 1.2 Procedure: 1.3 Tips:

Protocol for Ligation of Insert DNA into Plasmid Vector DNA

Materials:

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:

a. Linearized vector DNA: around 100ng

b. Insert DNA (at 3:1 molar excess over vector): variable

c. 10x ligation buffer: 1uL

d. T4 DNA Ligase: 1uL

e. ddwater: Rest of volume

Total volume: 10 uL

3. Vortex and spin briefly to collect drops.

4. Incubate the mixture at 16 degree for 45-60min.

5. Use the ligation mixture for transformation.

Tips:

• Thoroughly mix the 10x ligation buffer before use.

• The optima l insert/vector molar ratio is 3:1.

• To minimize recircularization of the cloning vector, dephosphorylate linearized plasmid DNA with Alkaline Phospha tase(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.