Team:Paris Saclay/digestion ligation

From 2013.igem.org

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(→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="33%" |
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| width="20%" |
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Tampon
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Buffer
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| width="34%" |
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| width="20%" |
Fast Digest  
Fast Digest  
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| width="33%" |
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| width="20%" |
Fast Digest  
Fast Digest  
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| width="34%" |
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| width="20%" |
Fast Digest  
Fast Digest  
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| width="33%" |
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| width="20%" |
Fast Digest  
Fast Digest  
|-
|-
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| width="33%" |
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| width="20%" |
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site
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Site
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| width="34%" |
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| 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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|}
|}
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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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|+  
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! scope=col | volume
! scope=col | volume
|-
|-
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| width="33%" |
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| width="20%" |
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Tampon FD 10X       
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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
|-
|-
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| 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%" |
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h20
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H20
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| width="34%" |
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| width="20%" |
14µL
14µL
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-
 
-
 
-
 
|}
|}
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Simple Digestion :  
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'''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%" |
-
Tampon FD 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%" |
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h20
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H20
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| width="34%" |
+
| width="20%" |
15.5µL
15.5µL
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|}
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1. Incubate at 37°C for 10-90min
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2. Inactivate digestion of enzymes by ethanol precipitation
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|}
 
 +
'''Ligation:'''
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1 .Incubate at 37°C during 10-90min
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Ligation is the method for the joining of nucleic acid fragments throught the action of enzyme.
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2. Inactivate digestion enzyms with ethanol precipitation
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Ligation:
 
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Mix in a molar ratio the insert and the linear vector
 
 +
Protocol
 +
Set up the following reaction in a microcentrifuge tube on ice.
 +
(T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)
 +
COMPONENT 20 μl REACTION
 +
* 10X T4 DNA Ligase Buffer* 2 μl
 +
* Vector DNA (3 kb) 50 ng (0.025 pmol)
 +
* Insert DNA (1 kb) 50 ng (0.076 pmol)
 +
* Nuclease-free water to 20 μl
 +
* T4 DNA Ligase 1 μl
 +
 +
Gently mix the reaction by pipetting up and down and microfuge briefly.
 +
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
 +
For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
 +
Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

Latest revision as of 14:59, 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:

Ligation is the method for the joining of nucleic acid fragments throught the action of enzyme.


Protocol

Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.) COMPONENT 20 μl REACTION

  • 10X T4 DNA Ligase Buffer* 2 μl
  • Vector DNA (3 kb) 50 ng (0.025 pmol)
  • Insert DNA (1 kb) 50 ng (0.076 pmol)
  • Nuclease-free water to 20 μl
  • T4 DNA Ligase 1 μl

Gently mix the reaction by pipetting up and down and microfuge briefly. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.



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