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From 2012.igem.org

Ligation Procedure

A reaction mixture was prepared containing,

Element	Volume
Upstream Digestion part	2 µL
Downstream Digestion part	2 µL
Destination Plasmid	2 µL
10X T4 DNA ligase buffer	2 µL
T4 DNA ligase	1 µL
H2O	11 µL

The mixtures were incubated for 10 minutes and then heat inactivated at 80°C for 20 minutes.

2 µL of the ligation product was transformed into 50 µL of competent E. coli, the transformants were then incubated overnight.