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Team:Tuebingen/Notebook/Protocols/preparative-restriction
From 2013.igem.org
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| - | <p>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. Heat inactivate phosphatase at 70 °C for 5 min. Proceed with ligation (3A-Assembly).</p> | + | <p>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. Proceed with ligation (3A-Assembly).</p> |
</div> | </div> | ||
Revision as of 01:21, 4 October 2013
Preparative Restriction Digest
Back to Protocols
Digestion
Reagents
| 10.0 µL | 10x NEBuffer 2 |
| 1.0 µL | 100x BSA |
| 5 - 10 µg | Plasmid DNA |
| 2.0 µL | EcoRI or XbaI |
| 2.0 µL | PstI or SpeI |
| to 100 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. Proceed with ligation (3A-Assembly).
