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

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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
  • 0,056g 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

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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

    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

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    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

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    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

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    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

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    Probe insertion

    Preparation of the plasmid backbone

    Materials
  • 2,5µg Plasmid DNA
  • 50U EcoRI (2,5µL@20 000U/mL)
  • 5µL CutSmart buffer 10X
  • 50µL Distilled Water
  • 3U CIAP (Calf Intestinal Alkaline Phosphatase, 3µL@ 1U/µL)
  • 1200 U T4 DNA Ligase (3µL@400 000/mL)e
  • 6µL T4 DNA Ligase Reaction Buffer 10X

    • Methods
    1. Plasmid Digestion
      1. Mix the plasmid DNA, EcoRI, the CutSmart buffer and distilled water until 50µL final volume
      2. Incubate 15 minutes at 37°C
    2. Serial dephosphorylations
      1. In each sample to dephosphorylate: add 1U (1µL) of CIAP
      2. Incubate 5 minutes at 37°C
      3. Incubate 15 minutes at 65°C (for inactivation of the phosphatase)
        Step is repeated three times (to ensure the avoidance of self-ligation).
        NB: At each step, sample is divided in two: the part that goes through a new dephosphorylation and the one that does not.
    3. Control by ligation (without the probe) and bacterial transformation
      1. Prepare 1uL (50ng) of the digested and X-times dephosphorylated plasmid
      2. Add 1uL of ligase, 2uL of its buffer and distilled water sat 20µL
      3. Incubate 10 minutes at Room Temperature
      4. Incubate 10 minutes at 65°C for inactivation
      5. Transformation (cf. upstream protocol)


    Preparation of the probe

    Materials
  • 300ng Probe DNA (1µL@315ng/µL)
  • 10U EcoR1 (0,5µL @20 000U/mL)
  • 2µL CutSmart Buffer 10X
  • 5Distilled water sat 20µL

    • Methods : Probe digestion
    1. Mix the probe DNA, EcoRI, the CutSmart Buffer and the distilled water until 20µL final volume
    2. Incubate 15 minutes at 37°C
    3. Incubate 20 minutes at 65°C for inactivation


    Ligation Probe - Plasmid backbone

    Materials
  • 12µL of the plasmid DNA digested and dephosphorylated 3 times
  • 4,7µL of the probe digested
  • 5µL T4 DNA Ligase (@400 000/mL)
  • 12µL T4 DNA Ligase 10X
  • 120µL distilled water

    • Methods
    1. Insertion probe
      1. Preheat tubes probe and plasmid backbone at 70°C to limit the hydrogen bounds
      2. Prepare the following mix (help: NEBBioCalculator)
        3. Molar ratio probe/plasmid 0:11:13:17:110:13:1 negative control without ligase
        Plasmid (dephosphorylated 3 times @50ng/µL)2µL
        Probe (@15,7ng/µL)00,3µL0,6µL1,3µL1,9µL0,6µL
        T4 DNA ligase (@400 000/mL)1µL0
        Buffer T4 Ligase 10X2µL
        Distilled WaterComplete to 20µL final volume
      3. Incubate 10 minutes at Room Temperature
      4. Incubate 10 minutes at 65°C for inactivation
    2. Transformation
        3. Transform 1µL of each product of ligation in 20µL competent DH5α.
        See protocol upstream.


    Screening

    Materials
  • 75mL LB+cam media
  • 15 miniprep columns (Monarch® Plasmid Miniprep Kit (NEB)
  • 15µL EcoR1 (@20 000U/mL)
  • 30µL Cutsmart buffer 10X
  • 300 µL distilled water
  • 2g agarose
  • 600mL TAE 1X
    • Methods
    1. Pick up 3 colonies per conditions of ligation (ratio 1:1 to 7:1 + neg control w/o ligase) to put them separately in 5mL LB+cam
    2. Amplify via overnight culture (37°C, agitation)
    3. Extract DNA from each cultures
    4. Digest 1µg of each DNA extract with EcoR1 (20U enzyme, 2µL CutSmart Buffer 10X, water until 20µL final volume and incubation 15’ at 37°C)
    5. Prepare a 2% agarose gel (2g agarose for 100mL TAE 1X) (see protocol above)
    6. Migrate the digested DNAs (see protocol above)
    7. Check which plasmid backbone inserted the probe (should appear at 96bp)

    CLOSE

    Plasmid activation

    Materials
  • 2µg detector (plasmid+probe)
  • 20U BmtI (2uL@10 000U/mL)
  • 20U BglI (2µL@10 000U/mL)
  • 20U Nt.BspQI (2uL@10 000U/mL)
  • 20U Nb.BtsI (2uL@10 000U/mL)
  • 4µL NEBuffer 3.1. 10X
  • 5µL CutSmart Buffer 10X
  • 29,4µL Water

    • Methods
    1. Prepare 2µg of backbone DNA in a tube
    2. Add 2µL 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 Nb.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

    Detection of the target

    Materials
  • 300ng activated detector
  • 300ng target DNA
  • 30µL Tris HCl Buffer 0,01M Ph=7,45

    • Methods
    1. Pre-warm the target at 70°C during >10min.
    2. Prepare the following mixes
      3. Detector100ng100ng100ng
      Target0ng100ng200ng
      Tris HCl Buffer 0,01M pH=7,45Until 10µLUntil 10µLUntil 10µL
    3. Incubate the tube at 70°C during 8 minutes
    4. Use 5µL of each mix to transform competent DH5a (see protocol above)

    CLOSE

    Purification and DNA extraction from agarose gel

    Were carried out according to the NucleoSpin Gel® (MN) and PCR Clean-up Kits (Invitrogen)

    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

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    Bacteria lyophilisation and resuspension

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

    • Methods
    NB : Work under Microbiological Safety Bench and on ice
    1. Lyophilize the bacteria
    2. Conserve at 4°C
    3. Resuspend the lyophilised bacteria with 500uL of the resuspension solution
    4. Use or conserve the bacteria at -20°C

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