Team:INSA-Lyon/Protocols/Digestio

From 2010.igem.org

Protocols

1. Competent cells
2. Transformation
3. DNA extraction
4. Digestion
5. Ligation
6. Measure of temperature and shaking speed influence
7. Measure of osmotic pressure influence
8. Granules extraction and intein cleavage
9. Biofilms quantification
10. Extra

    Contents 1 Digestion 1.1 Single digestion 1.2 Double digestion EcoRI/SpeI: 1.3 Double digestion EcoRI/XbaI: 1.4 Double digestion XbaI/PstI: 1.5 Double digestion XbaI/SpeI:

    Digestion

    
    

    Single digestion

    1. Add x µL DNA (0,5 to 1 ug)
    2. Add 15 µL DNA extracted from the part transformation.
    3. Add 2 µL 10X buffer
    4. Add the enzyme last
    5. Add 0,5 to 2 µL restriction enzyme (max 1/10 of final volume)
    6. Add sterile water qs 20 µL
    7. Incubate 1h at 37°C.

    
    

    Double digestion EcoRI/SpeI:

    1. Add x µL DNA (0,5 to 1 µg). Add 15 µL DNA extracted from the part transformation.
    2. Add 2 µL buffer Tango 10x
    3. Add 1 µL SpeI
    4. Add sterile water qs 20 µL
    5. Incubate 1h at 37°C
    6. Add 2 µL buffer Tango 10x
    7. Add 1 µL EcoRI
    8. Incubate 1h at 37°C

    Double digestion EcoRI/XbaI:

    1. Add x µL DNA. Add 15 µL DNA extracted from the part transformation.
    2. Add 4 µL buffer Tango 10x
    3. Add 1 µL XbaI
    4. Add 0,5 µL EcoRI
    5. Add sterile water qs 20 µL
    6. Incubate 1h at 37°C

    Double digestion XbaI/PstI:

    1. Add x µL DNA. Add 15 µL DNA extracted from the part transformation.
    2. Add 2 µL buffer Tango 10x
    3. Add 1µL XbaI
    4. Add 1 µL PstI
    5. Add sterile water qs 20 µL
    6. Incubate 1h at 37°C

    Double digestion XbaI/SpeI:

    1. Add x µL DNA. Add 15 µL DNA extracted from the part transformation.
    2. Add 2 µL buffer Tango 10x
    3. Add 1 µL XbaI
    4. Add 1 µL SpeI
    5. Add sterile water qs 20 µL
    6. Incubate 1h at 37°C
    Retrieved from "http://2010.igem.org/Team:INSA-Lyon/Protocols/Digestio"