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Team:University College London/Protocols/Week13/2
From 2012.igem.org
Revision as of 22:50, 26 September 2012 by Erinoerton (Talk | contribs)
Ligations for irrE, nuclease, laccase & curli
Digest upstream part with EcoRI-HF™ and SpeI
| Constitutive promoter-rbs construct | 500 ng |
|---|---|
| EcoRI-HF | 1µl |
| Spel | 1 µl |
| 10X NEBuffer 2 | 5 µl |
| 100X BSA | 0.5 µl |
| H2O | to 50 µl |
Digest downstream part with Xbal and Pstl
| Curli (from PCR product) Laccase (from PCR product) irrE (from PCR product) 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 |
| PSB1C3 | 500 ng | |
|---|---|---|
| EcoRI-HF | 1 µl | |
| Pstl | 1 µl | |
| 10X NEBuffer 2 | 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.
| 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:
| 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 |
