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From 2012.igem.org

Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes. Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. {| class="bigtable"

Destination Plasmid DNA	500 ng
EcoRI-HF	1 µl
PstI	1 µl
10X NEBuffer	2.5 µl
100X BSA	0.5 µl
H2O	to 50 µl
Upstream Part digestion	2 µl
Destination Plasmid digestion	2 µl
10X T4 DNA Ligase Buffer	2 µl
T4 DNA Ligase	2 µl
H2O	11 µl