Team:EPF-Lausanne/Protocols/Digestion

From 2009.igem.org

(Difference between revisions)
 
(5 intermediate revisions not shown)
Line 15: Line 15:
<html><center>
<html><center>
-
<font size="6" color="#007CBC"><i>Digestion Protocol</i></font>  
+
<font size="6" color="#007CBC">Digestion Protocol</font>  
</center></html>
</center></html>
<br>
<br>
----
----
-
<br>
 
-
<br>
 
-
 
Line 27: Line 24:
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.
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==
 +
<center>
{| class="wikitable" style="text-align:left; width:50%;"
{| class="wikitable" style="text-align:left; width:50%;"
-
|+ '''<big>Reaction Mix</big>'''
 
|-
|-
! scope=row | DNA
! scope=row | DNA
Line 51: Line 50:
|'''10 µl'''
|'''10 µl'''
|}
|}
-
 
+
</center>
-
+
-
+
-
Dephosphorylation
+
==Dephosphorylation==
 +
 
Add 1 µl of artic phosphatase to the vector to prevent ligation of the vector on itself.
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
+
==Incubation==
-
EcoRI 100 100 100 100 optional 20min @ 65°C
+
 
-
SpeI 75 100 25 100 X 20min @ 80°C
+
:Vector: 2h at 37°C → add artic phophatase → 20min at 37°C
-
XbaI 0 100 75 100 X 20min @ 65°C
+
:Insert: 1h20min at 37°C
-
PstI 75 75 100 50 X 20min @ 80°C
+
 
-
NotI 0 50 100 25 X 20min @ 65°C
+
==Enzyme properties - ''www.neb.com''==
 +
 
 +
{| class="wikitable" style="text-align:center; width:80%;"
 +
|-
 +
! scope=col |
 +
! scope=col | NEB 1  
 +
! scope=col | NEB 2
 +
! scope=col | NEB 3  
 +
! scope=col | NEB 4  
 +
! scope=col | BSA
 +
! scope=col | Inactivation
 +
|-
 +
! scope=row | EcoRI
 +
|100
 +
|100
 +
|100
 +
|100
 +
|optional
 +
|20 min at 65°C
 +
|-
 +
! scope=row | SpeI
 +
|75
 +
|100
 +
|25
 +
|100
 +
|X
 +
|20 min at 80°C
 +
|-
 +
! scope=row |XbaI
 +
|0
 +
|100
 +
|75
 +
|100
 +
|X
 +
|20 min at 65°C
 +
|-
 +
! scope=row | PstI
 +
|75
 +
|75
 +
|100
 +
|50
 +
|X
 +
|20 min at 80°C
 +
|-
 +
! scope=row | NotI
 +
|0
 +
|50
 +
|100
 +
|25
 +
|X
 +
|20 min at 65°C
 +
|}
</div><div CLASS="epfl09bouchon"></div>
</div><div CLASS="epfl09bouchon"></div>

Latest revision as of 18:45, 20 October 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: 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


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