Team:INSA-Lyon/Protocols/Digestio
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Revision as of 20:39, 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
Digestion
Single digestion :
- Add x µL DNA (0,5 to 1 ug)
- Add 15 µL DNA extracted from the part transformation.
- Add 2 µL 10X buffer
- Add the enzyme last
- Add 0,5 to 2 µL restriction enzyme (max 1/10 of final volume)
- Add sterile water qs 20 µL
- Incubate 1h at 37°C.
Double digestion EcoRI/SpeI:
- Add x µL DNA (0,5 to 1 µg). Add 15 µL DNA extracted from the part transformation.
- Add 2 µL buffer Tango 10x
- Add 1 µL SpeI
- Add sterile water qs 20 µL
- Incubate 1h at 37°C
- Add 2 µL buffer Tango 10x
- Add 1 µL EcoRI
- Incubate 1h at 37°C
Double digestion EcoRI/XbaI :
- Add x µL DNA. Add 15 µL DNA extracted from the part transformation.
- Add 4 µL buffer Tango 10x
- Add 1 µL XbaI
- Add 0,5 µL EcoRI
- Add sterile water qs 20 µL
- Incubate 1h at 37°C
Double digestion XbaI/PstI :
- Add x µL DNA. Add 15 µL DNA extracted from the part transformation.
- Add 2 µL buffer Tango 10x
- Add 1µL XbaI
- Add 1 µL PstI
- Add sterile water qs 20 µL
- Incubate 1h at 37°C
Double digestion XbaI/SpeI :
- Add x µL DNA. Add 15 µL DNA extracted from the part transformation.
- Add 2 µL buffer Tango 10x
- Add 1 µL XbaI
- Add 1 µL SpeI
- Add sterile water qs 20 µL
- Incubate 1h at 37°C