Team:HKUST/Protocols/Ligation

Ligation

Purpose: To ligate desired insert DNA fragment into vector with specific restriction site.

Materials: digested insert DNA and plasmid DNA with the same restriction site, T4 DNA ligase, 10X T4 Ligation Buffer, CIP (Calf Intestinal Alkaline Phosphatase), ddH2O

Procedure:

a.Add sufficient water to bring to a total volume of 10 μL or 20 μL, depending on the volume of the two DNA pieces.
b.Add 50ng of vector DNA.
c.Add enough amount of insert DNA, which can be calculated by ligation calculator online.
d.Add 2μL ligation buffer and 0.5μL ligase.
e.Incubate at 16 °C overnight or at room temperature for 8 hours.

Tips:

A. Ligase blank control group is recommended.
B. The plasmid DNA and insert DNA for ligation must be added with CIP when enzyme digestion.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing