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From 2012.igem.org

The first digest was carried out in order to prepare the Upstream Part for ligation through an EcoRI-HF™ and SpeI preparative digest:

Ingredient	Amount
Upstream Part Plasmid	500 ng
EcoRI-HF	1 µl
Spel	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Another digest was carried out in order to prepare the downstream DNA for ligation through an XbaI/PstI preparative digest:

Ingredient	Amount
Downstream Part Plasmid	500 ng
Xbal	1 µl
Pstl	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Another digest was carried out to prepare the backbone plasmid through an EcoRI-HF/PstI preparative digest:

Ingredient	Amount
Destination Plasmid	500 ng
EcoRI-HF	1 µl
PstI	1 µl
10X NEBuffer	2.5 µl
100X BSA	0.5 µl
H2O	to 50 µl

For each of the digests above, 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, using the following:

Ingredient	Amount
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

After this is prepared, incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.