Team:INSA-Lyon/Protocols/Digestio

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Revision as of 21:31, 24 August 2010




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Thank you Groningen for sharing your code, you've been most helpful !

Protocols

Currently under construction



  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






    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





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