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Team:INSA-Lyon/Protocols/Ligation
From 2010.igem.org
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Revision as of 20:41, 25 August 2010
Protocols
Currently under construction
- Competent cells
- Transformation
- DNA extraction
- Digestion
- Ligation
- Measure of temperature and shaking speed influence
- Measure of osmotic pressure influence
- Granules extraction and intein cleavage
- Biofilms quantification
- Extra
Ligation
- Add x µL DNA (the entire digestion gel purification for part ligation) Plasmid: 50 to 200 ng; Insert: 100 ng (0,5 kb) to 1 µg (10 kb))
- Add 2 µL Buffer T4 DNA ligase (Attention: this buffer contains ATP, defrost on ice)
- Add 0,5 µL T4 DNA ligase (0,5 U) --> Add the enzyme last
- Add sterile water qs 20 µL
If it follows a digestion without purification: thermoinactivation 20 min at 70°C
Incubate 3h at room temperature or overnight at 15°C
