Team:Paris Saclay/digestion ligation

From 2013.igem.org

(Difference between revisions)
(→Digestion and Ligation)
(→Digestion and Ligation)
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Digestion :  
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'''Digestion :'''
{| class="wikitable centre" width="80%"
{| class="wikitable centre" width="80%"
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! scope=col | XbaI
! scope=col | XbaI
|-
|-
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| width="30%" |
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| width="20%" |
Buffer  
Buffer  
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| width="30%" |
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| width="20%" |
Fast Digest  
Fast Digest  
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| width="30%" |
+
| width="20%" |
Fast Digest  
Fast Digest  
-
| width="30%" |
+
| width="20%" |
Fast Digest  
Fast Digest  
-
| width="30%" |
+
| width="20%" |
Fast Digest  
Fast Digest  
|-
|-
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| width="33%" |
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| width="20%" |
Site
Site
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| width="34%" |
+
| width="20%" |
5'-G/AATTC-3'
5'-G/AATTC-3'
3'-CTTAA/G-5'
3'-CTTAA/G-5'
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| width="33%" |
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| width="20%" |
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5'CTGCA/G 3'
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5'-CTGCA/G-3'
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3'G/ACGTC 5'
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3'-G/ACGTC-5'
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| width="34%" |
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| width="20%" |
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5'A/CTAGT 3'
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5'-A/CTAGT-3'
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3'T G ATC/A5'
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3'-TGATC/A-5'
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| width="33%" |
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| width="20%" |
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5'T/CTAGA3'
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5'-T/CTAGA-3'
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3'AGATC/T5'
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3'-AGATC/T-5'
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-
 
+
-
 
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|}
|}
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Double Digestion :  
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'''Double Digestion :'''
{| class="wikitable centre" width="80%"
{| class="wikitable centre" width="80%"
|+  
|+  
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! scope=col | volume
! scope=col | volume
|-
|-
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| width="33%" |
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| width="20%" |
Buffer Fast Digest 10X       
Buffer Fast Digest 10X       
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| width="34%" |
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| width="20%" |
3µL
3µL
|-
|-
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| width="33%" |
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| width="20%" |
Enzyme 1               
Enzyme 1               
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| width="34%" |
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| width="20%" |
1.5 μl
1.5 μl
|-
|-
-
| width="33%" |
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| width="20%" |
Enzyme 2
Enzyme 2
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| width="34%" |
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| width="20%" |
1.5 μl
1.5 μl
|-
|-
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| width="33%" |
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| width="20%" |
-
h20
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H20
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| width="34%" |
+
| width="20%" |
14µL
14µL
-
 
-
 
-
 
-
 
|}
|}
-
Simple Digestion :  
+
'''Single Digestion :'''
{| class="wikitable centre" width="80%"
{| class="wikitable centre" width="80%"
|+  
|+  
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! scope=col | volume
! scope=col | volume
|-
|-
-
| width="33%" |
+
| width="20%" |
Buffer Fast Digest 10X       
Buffer Fast Digest 10X       
-
| width="34%" |
+
| width="20%" |
3µL
3µL
|-
|-
-
| width="33%" |
+
| width="20%" |
Enzyme               
Enzyme               
-
| width="34%" |
+
| width="20%" |
1.5 μl
1.5 μl
|-
|-
-
| width="33%" |
+
| width="20%" |
-
h20
+
H20
-
| width="34%" |
+
| width="20%" |
15.5µL
15.5µL
 +
|}
 +
1. Incubate at 37°C for 10-90min
 +
2. Inactivate digestion of enzymes by ethanol precipitation
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|}
 
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-
 
-
1 .Incubate at 37°C for 10-90min
 
-
2. Inactivate digestion of enzymes by ethanol precipitation
 
-
Ligation:  
+
'''Ligation:'''
Mix in a molar ratio the insert and the linear vector
Mix in a molar ratio the insert and the linear vector

Revision as of 14:46, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Digestion and Ligation

Digestion :

Enzyme EcoRI PstI SpeI XbaI

Buffer

Fast Digest

Fast Digest

Fast Digest

Fast Digest

Site

5'-G/AATTC-3' 3'-CTTAA/G-5'

5'-CTGCA/G-3' 3'-G/ACGTC-5'

5'-A/CTAGT-3' 3'-TGATC/A-5'

5'-T/CTAGA-3' 3'-AGATC/T-5'


Double Digestion :

DNA volume

Buffer Fast Digest 10X

3µL

Enzyme 1

1.5 μl

Enzyme 2

1.5 μl

H20

14µL


Single Digestion :

DNA volume

Buffer Fast Digest 10X

3µL

Enzyme

1.5 μl

H20

15.5µL


1. Incubate at 37°C for 10-90min

2. Inactivate digestion of enzymes by ethanol precipitation


Ligation:

Mix in a molar ratio the insert and the linear vector




Retrieved from "http://2013.igem.org/Team:Paris_Saclay/digestion_ligation"