Team:Grenoble-Alpes/Protocols

Lab

Protocols

Facing back to the Etendard glacier, in between the peaks of Maurienne and the ones of Oisan.
Credits: Estelle Vincent

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

    CLOSE

    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

    CLOSE

    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

    CLOSE

    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

    CLOSE

    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

    CLOSE

    CaCl2 100mM

    Materials
  • 0,056g CaCl2 (111g/mol)
  • 5mL Sterile Water

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

    CLOSE

    MgCl2 100mM

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

    • Methods
    1. Dissolve 0,610g of MgCl2 in 30mL of sterile water
    2. Conserve at 4°C

    CLOSE

    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

    CLOSE

    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

    CLOSE

    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

    CLOSE

    EXPERIMENTATIONS

    Competant bacteria

    Standard protocol

    Materials
  • 100 mL Bacterial Culture (DO=0,5-0,6)
  • 20mL MgCl2 (100mM)
  • 2mL CaCl2 (100mM) + Sucrose (100mM)
  • Glycérol 15%

    • Methods
    1. Centrifuge the culture at 5000rpm for 10 minutes at 4°C
    2. Remove the supernatant and resuspend pellet in 20mL of cold MgCl2
    3. Incubate 30 minutes on ice
    4. Centrifuge at 4000rpm for 10 minutes at 4°C
    5. Remove the supernatant and resuspend pellet in 2mL of CaCl2 and Glycérol 15%
    6. Aliquot 50uL in eppendorf previously cooled at -80°C
    7. Conserve at -80°C
    Protocol for lyophilisation

    Materials
  • 100 mL Bacterial Culture (DO=0,5-0,6)
  • 20mL MgCl2 (100mM)
  • 2mL CaCl2 (100mM) + Sucrose (100mM)
  • Sterile Ampoules

    • Methods
    1. Centrifuge the culture at 4000rpm for 10 minutes at 4°C
    2. Remove the supernatant and resuspend pellet in 20mL of MgCl2
    3. Centrifuge at 4000rpm for 10 minutes at 4°C
    4. Remove the supernatant and resuspend pellet in 2mL of CaCl2 + Sucrose
    5. Aliquot 500µL by sterile ampoules
    6. Conserve at -80°C

    CLOSE

    Electrophorese

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

    • Methods for a 1% agarose gel
    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
      Add Loading Dye 6X to dilute it until 1X (usually 2µL added to 10µL sample)
      NB: Maximum volume in the well is around 25µL & Minimum quantity of DNA detectable is around 25µg (for plasmid >3kb)
    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 to 1h protected from light
      4. Wash the gel in a tank of distilled water
      5. Read under UV

    CLOSE

    Bacterial Transformation

    Materials
  • 25uL DH5a Competent Cells
  • 1 to 5µL DNA (concentration>20ng/µL)
  • 450µL SOC
  • 2 Petri dish

    • Methods
    NB : Work under Microbiological Safety Bench and on ice
    1. Add DNA in 25µL of competent bacteria
    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

    CLOSE

    Miniprep, Maxiprep

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

    • CLOSE

    PCR

    Materials
  • 2,5µL Primers Forward (10µM)
  • 2,5µL Primers Reverse (10µM)
  • 25µL Polymerase Mix
  • 10ng Probe DNA (1µL)
  • Sterile Water sat (sufficient amount to) 50µL

    • 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

    CLOSE

    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

    CLOSE

    Plasmid activation

    Materials
  • 2µg Backbone DNA (plasmid+probe)
  • 4µL BmtI
  • 2µL BglI
  • 2µL Nt.BspQI
  • 2µL Nb.BtsI
  • 4µL NEBuffer 3.1. 10X
  • 5µL CutSmart Buffer 10X
  • 29,4µL Water

    • Methods
    1. Prepare 2µg of backbone DNA in a tube
    2. Add 4µL of BmtI with 2µL of NEBuffer 3.1. and 12,4µL of water
    3. Incubate 1 hour at 37°C
    4. Incubate 20 minutes at 65°C for inactivation
    5. Add 2µL of BglI with 1µL of NEBuffer 3.1. and 7µL of water
    6. Incubate 15 minutes at 37°C
    7. Add 2µL of Nt.BspQI with 1µL of NEBuffer 3.1. and 7µL of water
    8. Incubate 1 hour at 50°C
    9. Incubate 20 minutes at 80°C for inactivation
    10. Add 2µL of Nt.BtsI with 5µL of CutSmart Buffer and 3µL of water
    11. Incubate 1 hour at 37°C
    12. Incubate 20 minutes at 80°C for inactivation

    CLOSE

    Purification and DNA extraction from agarose gel

    Were carried out according to the NucleoSpin Gel® and PCR Clean-up Kits

    CLOSE

    Plasmid drying

    Materials
  • 15µL Plasmid
  • 15µL Sterile Water
  • Speed Vac

    • Methods
    1. Put 15µL of plamid in the Speed Vac
    2. Let it for 45 minutes
    3. Resuspended in 15uL of distillated water

    CLOSE

    Bacteria lyophilisation and resuspension

    Materials
  • 500µL Competent Bacteria
  • 500µL Resuspension Solution for Lyophilized Bacteria

    • Methods
    NB : Work under Microbiological Safety Bench and on ice
    1. Lyophilize the bacteria coming from the protocol “Competent Bacteria for Lyophilisation”
    2. Conserve at 4°C
    3. Resuspension
      1. Resuspend the lyophilised bacteria with 500uL of the resuspension solution
      2. Incubate 10 minutes on ice
    4. Use or conserve the bacteria at -20°C

    CLOSE

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