Team:Grenoble-Alpes/Protocols

Lab

Protocols

Preparations

LB Broth

Materials
  • 20g LB Broth Base (powder)
  • 1L Distillated Water

    • Methods
    1. Add 20g of LB Broth Base per 1L of distillated water
    2. Homogenize
    3. Autoclave at 121°C for 15 minutes

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    LB Agar

    Materials
  • 32g LB Agar (powder)
  • 1L Distilled Water

    • Methods
    1. Add 32g of LB Agar per 1L of distilled water
    2. Homogenize
    3. Autoclave at 121°C for 15 minutes

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    Chloramphenicol (Cam) 25ug/mL Stock

    Materials
  • 0,25g Chloramphenicol (25mg/mL)
  • 10mL Ethanol

    • Methods
    1. Add 0,25g of Cam per 10mL of ethanol
    2. Homogenize by vortexing
    3. Filter with a 22mm membrane filter
    4. Conserve at -20°C in 1mL aliquot

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    Petri Dish Preparation

    Materials
  • 20mL LB Agar
  • 20µL Cam (25µg/mL)

    • Methods
    1. Add of 20uL Cam in 20mL of liquid LB Agar
    2. Pour the solution in the plate
    3. Wait until the agar solidifies
    4. Conserve returned in 4°C

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    Glycerol 80%

    Materials
  • 80mL Glycerol 100%
  • 20mL Sterile Water

    • Methods
    1. Harvest 80mL of glycerol 100%
    2. Add 20mL of sterile water
    3. Conserve at 4°C

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    CaCl2 100mM

    Materials
  • 0,0555g CaCl2 (111g/mol)
  • 5mL Sterile Water

    • Methods
    1. Dissolve 0,056g of CaCl2 in 5mL of sterile water
    2. Conserve at 4°C

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    MgCl2 100mM

    Materials
  • 0,610g MgCl2 (203,31g/mol)
  • 30mL Sterile Water

    • Methods
    1. Dissolve 0,6105g of MgCl2 in 30mL of sterile water
    2. Conserve at 4°C

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    Sucrose 100mM

    Materials
  • 0,171g Sucrose
  • 5mL Sterile Water

    • Methods
    1. Dissolve 0,171g of sucrose in 5mL of sterile water
    2. Conserve at 4°C

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    IPTG 4mM

    Materials
  • 80µL IPTG (500mM)
  • 10mL LB
  • 10µL Cam (25ug/mL)

    • Methods
    1. Add 10µL of Cam in 10mL of LB
    2. Homogenize
    3. Remove 90µL of solution and add 80µL of IPTG
    4. Conserve at -20°C

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    Resuspension Solution for Lyophilized Bacteria

    Materials
  • 350µL DMSO
  • 1,5µL 2-mercaptoethanol 1%
  • 5mL Sterile Water

    • Methods
    1. Add 350µL of DMSO and 1,5µL of 2-mercaptoethanol in 5mL of sterile water
    2. Conserve at 4°C

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    Experimentations

    Electrophorese

    Materials
  • 1g Agarose (powder)
  • 100mL TAE
  • Distilled Water
  • 25µL Gel Red

    • Methods
    1. Gel preparation
      1. Dissolve 1g of agarose in 100mL of TAE (1X)
      2. Pour the solution into the gel mould
      3. Let the solution gel (almost 15 minutes)
    2. Preparation of the samples to deposit
    3. The migration is done at 100V during 30 minutes
    4. Revelation
      1. Prepare 250mL of distilled water in a tank
      2. Add 25uL of Gel Red. Do not forget to use gloves to manipulate the Gel Red
      3. Incubate the gel in the solution for 10 minutes at the obscurity
      4. Wash the gel in a tank of distilled water

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    Bacterial Transformation

    Materials
  • 25uL DH5a Competent Cells
  • 1,5µL DNA
  • 450µL SOC
  • 2 Petri dish

    • Methods
    NB : Work under Microbiological Safety Bench and on ice
    1. Add 1,5µL of DNA in 25µL
    2. Gently invert the tube 4-5 times to mix cells and DNA. Do not vortex
    3. Incubate 30 minutes on ice
    4. Heat shock at 42°C for 1 minutes. Do not mix
    5. Place on ice for 2 minutes. Do not mix
    6. Incubate at 37°C and 150rpm for 2 hours
    7. Mix the cells thoroughly by inverting the tube
      1. Deposit 50µL on the first plate
      2. Centrifuge at 10000rpm for 3 minutes at Roof Temperature
      3. Remove a part of the supernatant and resuspend pellet with the rest
      4. Deposit the mixing on the second plate
    8. Incubate overnight at 37°C with plates upside down

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    Miniprep, Maxiprep

  • Minipreps were carried out according to the Monarch® Plasmid Miniprep Kit
  • Maxipreps were carried out according to the Pure Link® HiPure Plasmid DNA Purification Kits

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    PCR

    Materials
  • 2,5µL Primers Forward (10µM)
  • 2,5µL Primers Reverse (10µM)
  • 25µL Polymerase Mix
  • 1µL Probe DNA
  • 19µL Sterile Water

    • Methods
    1. Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin
    2. Transfer reaction quickly to a preheated thermocycler at 98°C
    3. Thermocycling conditions
      1. Initial denaturation at 98°C during 30 seconds
      2. 35 Cycles : 10 seconds at 98°C - 30 seconds at 57°C - 30 seconds at 72°C
      3. Final extension at 72°C during 2 minutes
    4. Conserve at -20°C

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    Probe insertion

    Materials
  • 2,5µL Plasmid DNA
  • 1,8µL EcoRI
  • 7µL EcoRI Buffer
  • 88µL Distilled Water
  • 1µL CIAP
  • 2µL T4 DNA Ligase
  • 4µL T4 DNA Ligase Reaction Buffer (10X)
  • 1µL Probe DNA

    • Methods
    1. EcoRI Plasmid Digestion
      1. Mix the plasmid DNA, 1,3µL of EcoRI, 5µL of buffer and 40,7µL of distilled water
      2. Incubate 15 minutes at 37°C
    2. Dephosphorylation
      1. Add 1µL of CIAP
      2. Incubate 5 minutes at 37°C
      3. Incubate 15 minutes at 65°C
        NB : This steep can be repeated until the plasmid is not ligature on itself
      4. Control by ligation and bacterial transformation
        1. Prepare 1µL of the digested and dephosphorylated plasmid
        2. Add 1µL of ligase and 2µL of its buffer and 16µL of distilled water
        3. Incubate 10 minutes at Roof Temperature
        4. Incubate 10 minutes at 65°C for inactivation
        5. Transformation (cf. protocols)
          NB : This steep is repeated in order to have the minimum of colonies
    3. EcoRI Probe Digestion
      1. Mix 1µL of probe DNA, 0,5µL of EcoRI, 2µL of CutSmart Buffer and 16,5µL of distilled water
      2. Incubate 15 minutes at 37°C
      3. Incubate 20 minutes at 65°C for inactivation
    4. Ligature
      1. Preheat tube at 70°C to limit the hydrogen bonded
      2. Add 0,3µL of the preparation containing the probe, 2µL of the dephosphorylated plasmid (100ng), 1µL of ligase, 2µL of its buffer and 15µL of distilled water
      3. Incubate 10 minutes at Roof Temperature
      4. Incubate 10 minutes at 65°C for inactivation

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