Team:University College London/Week12Joanne3

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