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Team:Tuebingen/Notebook/Protocols/3a-assembly
From 2013.igem.org
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<li><a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/trafo">Transform</a> 3A-Assembly product in cells.</li> | <li><a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/trafo">Transform</a> 3A-Assembly product in cells.</li> | ||
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Latest revision as of 12:11, 4 October 2013
3A-Assembly
Digestion
Reagents
| 2.5 µL | 10x NEBuffer 2 |
| 0.25 | 100x BSA |
| 250 - 300 ng | Plasmid DNA |
| 0.5 µL | EcoRI or XbaI |
| 0.5 µL | PstI or SpeI |
| to 25 µL | Aqua dest. |
| 1/10 vol | 10X Antarctic Phosphatase |
Procedure
- Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.
- On the next day, heat inactivate restriction enzymes at 80°C for 20 min.
- Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions.
- Heat inactivate phosphatase at 70 °C for 5 min.
- Perform the previous steps for upstream part plasmid, downstream part plasmid, and destination part plasmid. Digest upstream part with EcoRI / SpeI, downstream part with XbaI / PstI, and destination part plasmid with EcoRI / PstI.
Ligation
Reagents
| 2.0 µL | 10x T4 DNA Ligase Buffer |
| 2.0 | Upstream Part digestion |
| 2.0 | Downstream Part digestion |
| 2.0 | Destination Part digestion |
| 2.0 µL | T4 DNA Ligase |
| 11 µL | Aqua dest. |
Procedure
- Mix all reagents in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.
- On the next day, heat inactivate restriction enzymes at 80°C for 20 min.
- Transform 3A-Assembly product in cells.
