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Team:Paris Saclay/digestion ligation
From 2013.igem.org
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.
