"

From 2012.igem.org

Protocol 2: Ligations for ire, nuclease, laccase, curli

Digest Upstream Part with EcoRI-HF™ and SpeI

Ingredient	Amount
Constitutive promoter – rbs construct	500 ng
EcoRI-HF	1 µl
Spel	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Digest Downstream Part with Xbal and Pstl

Ingredient	Amount
Curli (from pcr product) Laccase (from pcr product) irrE (from pcr) Nuclease (in puc57)	500 ng
Xbal	1 µl
Pstl	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Ingredients	Amounts
EcoRI-HF	1 µl
Pstl	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

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.

Ingredients	Amounts
Upstream Part digestion	2 µl
Downstream Part digestion	2 µl
Destination Plasmid digestion	2 µl
10X T4 DNA Ligase Buffer	2 µl
T4 DNA Ligase	1 µl
H2O	11 µl

Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Summary of the cuts:

No.	Part	Enzymes
1	laccase	X+P
2	curli	X+P
3	irrE	X+P
4	irrE	E+P
5	nuclease	X+P
6	nuclease	X+P
7	plasmid backbone	E+P
8	Plasmid backbone	E+P
9	Linker	E+S