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Team:EPF-Lausanne/Protocols/Digestion
From 2009.igem.org
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.
| 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: 1h @ 37°C → add artic phophatase → 20min @ 37°C Insert: 1h20min @ 37°C
Enzyme properties - www.neb.com
NEB 1 NEB 2 NEB 3 NEB 4 BSA Inactivation EcoRI 100 100 100 100 optional 20min @ 65°C SpeI 75 100 25 100 X 20min @ 80°C XbaI 0 100 75 100 X 20min @ 65°C PstI 75 75 100 50 X 20min @ 80°C NotI 0 50 100 25 X 20min @ 65°C
