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From 2012.igem.org

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.