Team:Grenoble Alpes/Protocols

PyoBuster - Protocols

Protocols

Protocols' Description

Ampicillin

  • Materials
    • 500 mg Ampicillin
    • 5 mL Sterile Water
  • Methods
    1. Dissolve 500 mg of ampicillin in 5mL of sterile water
    2. Conserve at -20°C

Arabinose 10%

  • Materials
    • 1 g of arabinose
    • 10 mL Sterile Water
  • Methods
    1. Dissolve 1 g of Arabinose in 10mL of sterile water
    2. Conserve at 4°C

BHL

  • Materials
    • 1mg solid BHL (C4-HSL)
    • 5.84 mL PBS to obtain [1mM]
  • Methods
    1. Dissolve 1mg of BHL in 5.84mL of PBS
    2. Use immediately

CaCl2 100 mM

  • Materials
    • 0.056 g CaCl2 (111g/mol)
    • 5 mL Sterile Water
  • Methods
    1. Dissolve 0.056 g of CaCl2 in 5 mL of sterile water
    2. Conserve at 4°C

Competent bacteria

  • Materials
    • 4.2 mL CaCl2 at 100mM
    • 10 mL overnight bacterial preculture
  • Methods
    Throughout the protocol, keep the bacteria and the CaCl2 ice-cooled
    1. Cool both preculture and CaCl2 on ice for 30 minutes
    2. Centrifuge the preculture at 4°C, 1500rpm for 10 minutes
    3. Discard the supernatant and resuspend the pellet in 2mL of ice-cooled CaCl2
    4. Incubate on ice for 5 minutes
    5. Centrifuge at 4°C, 1500rpm for 10 minutes
    6. Discard the supernatant and resuspend a second time in 2mL of ice-cooled CaCl2
    7. Incubate one ice for 5 minutes
    8. Discard the supernatant and resuspend the pellet in 200µL of CaCl2
    Immediately use the competent E. coli Nissle
    Other bacteria can be stocked at -80°C with glycerol 80%

Digestion

  • Materials
    • DNA (1500-2000 ng/µL)
    • 2 µL Buffer (10X)
    • 1-2 µL Restriction enzymes
    • Sterile Water sat (quantum satis) 20µL
  • Methods
    1. Add components to a clean tube (20µL)
    2. Sterile Water sat (quantum satis) 50µL
    3. Incubate the reaction at digestion temperature (37°C) for 4 hour backbone / insert
    4. The digested DNA is ready for clean-up or gel extraction.
    5. Storage -20°C

DNA extraction

  • DNA extraction of agarose gel PCR and digestion were carried out according to the Zymoclean® Gel DNA Recovery Kit (OZYME) instructions.
  • DNA was eluted in 15µL of sterile distilled water and stock at -20°C.

DNA genome extraction

  • Genome extraction was carried out according to the NucleoSpin® Tissue (Macherey-Nagel) instructions.
  • DNA was eluted in 100µL of Elution Buffer water and stock at -20°C.

Electrophorese

  • Materials
    • Agarose powder
    • TAE: Tris, EDTA, Acetic acid
    • Distilled water
    • GelRed
  • Methods
    1. Dissolve agarose powder in the corresponding volume of TAE (1X)
    2. Pour the solution into the gel mould
    3. Let the solution polymerize
    4. Add Loading Dye 6X to dilute it until 1X
    5. The migration is done at 50 or 100V depending on the purpose of the migration (gel extraction, verification,...)
    6. The revelation is done in GelRed 3X
    7. Incubate the gel in the solution for 30 minutes protected from light
    8. Wash the gel in a tank of distilled water
    9. Reveal with the Gel Doc EZ System by Bio-RadⓇ

Glycerol 80%

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

LB

  • Materials
    • 25g LB-Medium
    • 1L Sterile Water
  • Methods
    1. Dissolve 25 g of LB-Medium in 1 L of water
    2. Autoclave at 121°C for 15min
    3. Conserve at RT

LB agar

  • Materials
    • 32g LB-Agar
    • 1L Sterile Water
  • Methods
    1. Dissolve 32g of LB-Agar in 1L of water
    2. Autoclave at 121°C for 15min
    3. Conserve at RT

Ligation

  • Materials
    • 1µL T4 DNA Ligase
    • 2 µL T4 DNA Ligase Buffer (10X)
    • Backbone (75 ng)
    • Insert (depending on ratios, calculated thanks to Ligation Calculator, NEBBioCalculatorⓇ)
    • Sterile Water sat (quantum satis) 20µL
  • Methods
    1. Add each reagent to a clean tube (20 µL)
    2. Incubate at 24°C for 20 min

Miniprep

  • Minipreps were carried out according to the ZR Plasmid Miniprep® ( NEB) instructions.
  • Plasmids were eluted in 50µL of sterile distilled water and stock at -20°C.

Midiprep

  • Midipreps were carried out according to the NucleoBond® Xtra Midi (Machery-Nagel) instructions.
  • Plasmids were eluted in 50µL of sterile distilled water and stock at -20°C.

PBS

  • Materials
    • 4 g of NaCl
    • 100 mg of KCl
    • 720 mg of Na2HPO4
    • 123 mg of KH2PO4
    • 500mL Sterile Water
  • Methods
    1. Dissolve all the reagents in 500mL of sterile water
    2. Conserve at RT°C

PCR

  • Materials
    • Primers Forward (0.5 µM)
    • Primers Reverse (0.5 µM)
    • dNTPs (200 µM)
    • 25 µL Polymerase Mix or Polymerase (0.05 units/µL) + Buffer (1X)
    • 1 µL DNA (10 ng)
    • Sterile Water sat (quantum satis) 50µL
  • Methods
    1. Add reagents to a clean tube (50 µL)
    2. Put the tube in a thermocycler:
      1. Initial denaturation at 94°C for at least 30 seconds
      2. During 30-35 cycles : 15 seconds at 94°C - 30 seconds at 50-65°C - 1 min per 1 kb at 72°C
      3. Final extension at 72°C during 5 minutes
    3. Conserve at -20°C

Petri dish

  • Materials
    • Molten LB-Agar
    • Petri dishes plates
    • Antibiotic
  • Methods
    1. Add the desired Antibiotic in the molten LB-Agar
    2. Gently mix by swirling
    3. Pour into plates
    4. Let plates cool
    • Inverts them and conserve them at 4°C

Transformation

  • Materials
    • 25µL DH5α competent cells
    • 1 to 5µL DNA (concentration>20ng/µL)
    • 100µL SOC
    • Petri dish
  • Methods
    1. Add DNA in 25µL of competent bacteria
    2. Incubate 30 minutes on ice
    3. Place on ice for 2 minutes
    4. Incubate at 37°C for 2 hours (Camp) or 30 minutes (Amp)
    5. Centrifuge at 10000rpm for 3 minutes at Room Temperature
    6. Remove a part of the supernatant and resuspend pellet with the rest
    7. Deposit the mixing on the plate
    8. Incubate overnight at 37°C with plate upside down

Ultra-competent cells transformation buffer

  • Materials
    • HEPES 10mM
    • CaCl215mM
    • KCl 250mM
    • MnCl2 55mM
    • KOH
    • Sterile distilled water
    • 0.2 micron sterile filter
  • Methods
    1. Dissolve in sterile water
    2. Balance at pH=6.2 with KOH
    3. Filter sterilize the solution
    4. Stock at 4°C

Ultra-competent bacteria

  • Materials
    • Ultra-competent cells transformation buffer
    • DMSO
    • 22°C 225rpm pre-culture at OD600nm=0.5
  • Methods
    1. Ice-cool for 30 minutes
    2. Centrifugate at 4100rpm, 4°C for 10 minutes
    3. Discard supernatant and resuspend in a third of initial volume of transformation buffer
    4. Incubate 15 minutes on ice
    5. Centrifugate at 4100rpm, 4°C for 10 minutes
    6. Discard supernatant
    7. Resuspend in a twelfth of initial volume of transformation buffer
    8. Add DMSO at a final concentration of 5%
    9. Incubate on ice for 10 minutes
    10. Aliquoote into precooled micro-tubes
    11. Immediately store at -80°C