Team:EPF-Lausanne/Protocols/Digestion

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Contents

Digestion Protocol



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