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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	1 µL
10X T4 DNA ligase buffer	2 µL
T4 DNA ligase	1 µL
H2O	12 µL

We incubated each ligation mix at 30°C for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.

4 µL of the ligation product was transformed into 100 µL of competent E. coli, the transformants were then incubated for one hour.