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Team:EPF-Lausanne/Protocols/Digestion
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Revision as of 18:44, 20 October 2009
We want to have all the DNA preps at 700 ng/µl, for the vector as for the insert. If the DNA preps are not concentrated enough, you can add more DNA and increase the volume of the reaction, respecting the buffers concentration (10x). Complete with dH2O if needed.
Reaction Mix
| DNA | 7 µl (700 ng of 100 ng/µl prep) |
|---|---|
| NEB buffer 10x | 1 µl |
| BSA 10x | 1 µl |
| Enzyme 1 | 0.5 µl |
| Enzyme 2 | 0.5 µl |
| dH2O | 0 µl |
| Total | 10 µl |
Dephosphorylation
Add 1 µl of artic phosphatase to the vector to prevent ligation of the vector on itself.
Incubation
- Vector: 2h at 37°C → add artic phophatase → 20min at 37°C
- Insert: 1h20min at 37°C
Enzyme properties - www.neb.com
| NEB 1 | NEB 2 | NEB 3 | NEB 4 | BSA | Inactivation | |
|---|---|---|---|---|---|---|
| EcoRI | 100 | 100 | 100 | 100 | optional | 20 min at 65°C |
| SpeI | 75 | 100 | 25 | 100 | X | 20 min at 80°C |
| XbaI | 0 | 100 | 75 | 100 | X | 20 min at 65°C |
| PstI | 75 | 75 | 100 | 50 | X | 20 min at 80°C |
| NotI | 0 | 50 | 100 | 25 | X | 20 min at 65°C |
