Team:EPF-Lausanne/Protocols/Digestion

From 2009.igem.org

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{| class="wikitable" style="text-align:center; width:80%;"
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|+ Titre du tableau
 
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! scope=col |
! scope=col |
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! scope=col | Titre col. A
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! scope=col | NEB 1
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! scope=col | Titre col. B
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! scope=col | NEB 2
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! scope=col | Titre col. C
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! scope=col | NEB 3
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! scope=col | Titre col. D
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! scope=col | NEB 4
 +
! scope=col | BSA
 +
! scope=col | Inactivation
|-
|-
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! scope=row | Titre ligne 1
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! scope=row | EcoRI
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|donnée L1-A
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|100
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|donnée L1-B
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|100
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|donnée L1-C
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|100
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|donnée L1-D
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|100
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|optional
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|20 min at 65°C
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|-
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! scope=row | Titre ligne 2
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! scope=row | SpeI
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|donnée L2-A
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|75
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|donnée L2-B
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|100
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|donnée L2-C
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|25
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|donnée L2-D
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|100
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|X
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|20 min at 80°C
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|-
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! scope=row |Titre ligne 3
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! scope=row |XbaI
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|donnée L3-A
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|0
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|donnée L3-B
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|100
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|donnée L3-C
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|75
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|donnée L3-D
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|100
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|X
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|20 min at 65°C
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|-
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! scope=row | Titre ligne 4
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! scope=row | PstI
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|donnée L4-A
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|75
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|75
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|donnée L4-C
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|100
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|donnée L4-D
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|50
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|X
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|20 min at 80°C
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|-
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! scope=row | NotI
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|0
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|50
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|100
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|25
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|X
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|20 min at 65°C
|}
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-
 
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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
 
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Revision as of 07:45, 4 September 2009



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: 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 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


Retrieved from "http://2009.igem.org/Team:EPF-Lausanne/Protocols/Digestion"