Team:Paris Saclay/digestion ligation

From 2013.igem.org

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'''Digestion :'''
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{| class="wikitable centre" width="80%"
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|+
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|-
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! scope=col | Enzyme
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! scope=col | EcoRI
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! scope=col | PstI
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! scope=col | SpeI
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! scope=col | XbaI
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|-
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| width="20%" |
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Buffer
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| width="20%" |
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Fast Digest
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| width="20%" |
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Fast Digest
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| width="20%" |
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Fast Digest
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| width="20%" |
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Fast Digest
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|-
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| width="20%" |
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Site
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| width="20%" |
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5'-G/AATTC-3'
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3'-CTTAA/G-5'
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| width="20%" |
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5'-CTGCA/G-3'
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3'-G/ACGTC-5'
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| width="20%" |
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5'-A/CTAGT-3'
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3'-TGATC/A-5'
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| width="20%" |
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5'-T/CTAGA-3'
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3'-AGATC/T-5'
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|}
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'''Double Digestion :'''
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{| class="wikitable centre" width="80%"
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|+
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|-
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! scope=col | DNA
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! scope=col | volume
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|-
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| width="20%" |
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Buffer Fast Digest 10X     
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| width="20%" |
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3µL
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|-
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| width="20%" |
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Enzyme 1             
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| width="20%" |
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1.5 μl
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|-
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| width="20%" |
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Enzyme 2
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| width="20%" |
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1.5 μl
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|-
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| width="20%" |
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H20
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| width="20%" |
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14µL
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|}
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'''Single Digestion :'''
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{| class="wikitable centre" width="80%"
 +
|+
 +
|-
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! scope=col | DNA
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! scope=col | volume
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|-
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| width="20%" |
 +
Buffer Fast Digest 10X     
 +
| width="20%" |
 +
3µL
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|-
 +
| width="20%" |
 +
Enzyme             
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| width="20%" |
 +
1.5 μl
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|-
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| width="20%" |
 +
H20
 +
| width="20%" |
 +
15.5µL
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|}
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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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 +
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'''Ligation:'''
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Ligation is the method for the joining of nucleic acid fragments throught the action of enzyme.
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 +
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Protocol
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Set up the following reaction in a microcentrifuge tube on ice.
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(T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)
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COMPONENT 20 μl REACTION
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* 10X T4 DNA Ligase Buffer* 2 μl
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* Vector DNA (3 kb) 50 ng (0.025 pmol)
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* Insert DNA (1 kb) 50 ng (0.076 pmol)
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* Nuclease-free water to 20 μl
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* T4 DNA Ligase 1 μl
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Gently mix the reaction by pipetting up and down and microfuge briefly.
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For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
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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).
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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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