Team:ZJU-China/n p 11.htm



11 Ligation:


11.1 Use fermentas T4 DNA Ligase. Follow the manufacturer’s protocols.


11.2 Set up the following reaction in a microcentrifuge tube on ice.

Linear vector DNA 20 - 100ng
Insert DNA 10:1 molar ratio over vector
10*T4 DNA Ligase Buffer 2 μl
T4 DNA Ligase 1 μl
Nuclease-free water To 20 μl
Total volume 20 μl

11.3 Incubate 30 min at 22℃.


11.4 Heat inactivation of T4 DNA ligase at 65℃ for 10 min. (not necessary)


11.5 Use 10 μl of the mixture for transformation of 100μl competent cell.