Team:Paris Saclay/digestion ligation

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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.