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Team:Tuebingen/Notebook/Protocols/3a-assembly
From 2013.igem.org
3A-Assembly
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Digestion
Reagents
| 2.5 µL | 10x NEBuffer 2 |
| 0.25 µL | 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.
Run gel and continue with gelextraction.
Ligation
