Difference between revisions of "Team:Paris Bettencourt/Protocols"

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Revision as of 09:11, 11 September 2015

Analytical digestion protocol

  • Prepare the following mix:
    • 2µL 10X Digestion buffer
    • 0.5µL Eco31I
    • 0.5µL BbsI
    • 2µL of DNA (200ng)
    • 15µL water
  • incubate 1h at 37°C

Annealing Protocol

  • Phosphorylation of the oligos
    • 5.6μL DNAse/RNAse free water
    • 6.0μL oligo 1 (10µM)
    • 6.0μL oligo 2 (10µM)
    • 2.0μL 10X T4 DNA ligase buffer
    • 0.4μL T4 PolyNucleotide Kinase
      • Total: 20μL
  • incubate 30min at 37°C
  • add 1μL of 1M NaCl
  • incube 5min at 95°C
  • let the mix cool down
  • use 2μL of the mix as a 10X solution

Chemical test for competent cells

  • 20-30 sec at 8-10 krpm for the DNA tube (from the kit).
  • Thow cell competent on ice. Label 2 ml eppendorf µtube for each concentration and put it on ice.
  • Add 1µl of DNA into each µtube.
  • Add 50µl of competent cell into each tube. Flick gently to mix. Incubate in ice for 30 minutes. Pre-heat waterbath at 42°C.
  • Heat-shock cell by placing in waterbath 1 minute. CAUTION : The top need to be close to the water level, but not immerge in.
  • Keep back the tube to ice for 5 minutes.
  • Add 200µl of SOC media, incubate 37°C for 2h. Prepare agar plate of the media that you want, and label it (3 plate for each concentration).
  • Add 20µl of each tube n the appropriate plate. Incubate 37°C for OVN (12-16h).

Electroporation

  • Thaw electrocompetent cells on ice
  • Add 2µL of ligation product or 0.5µL of native plasmid to the cells
  • Transfer the cells in an 0.2mm electroporation cuvette
  • put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF
  • add 200µL of LB right after pulsing
  • recover 2 hours at 37°C
  • plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C

Electrocompetent Cells

  • Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
  • Incubate until the the DO600 reach 0.5 to 0.7
  • Place the cultures on ice for 15 minutes
  • For the culture in cold sterile 50mL falcon tubes
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 50mL cold distilled water
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 25mL cold distilled water
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 12.5mL cold 10% glycerol
  • Centrifuge them for 10 minutes at 6000rpm
  • Throw the supernatant
  • Resuspend the cells in 5mL cold 10% glycerol
  • Make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C

Digestion

  • Prepare the following mix:
    • 4μL of Enzyme 1 Fast Digest
    • 4μL of Enzyme 2 Fast Digest
    • 4μL of FastAP
    • 12μL of Fast Digest buffer 10X
    • 1 to 3 μg of DNA
    • up to 120μL of water
  • mix by pipetting up and down
  • incube 10 to 20 minutes at 37°C
  • incube 10 minutes at 68°C to inactivate the enzymes

Lactobacillus plantarum electrocompetent cells

Electrocompetent Cells Preparation Protocol for Lactobacillus Plantarum
  • Inoculate 5ml MRS medium with L. plantarum freezer stock.
  • Grow overnight at 30°C without shaking.
  • Add 1.25g glycine to two flasks of 50ml MRS medium.
  • Shake to dissolve.
  • Add 1ml overnight culture to each flask (1:50 dlution).
  • Culture for ~3 hours at 37°C with shaking.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 25ml ice-cold DI water.
  • Repeat it.
  • Centrifuge for 5min at 4000g
  • Resuspend in 5ml 50mM EDTA.
  • Incubate on ice for 5 minutes
  • Add 25ml ice-cold DI water.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 25ml ice-cold DI water.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
  • Repeat it.
  • Centrifuge culture for 5min at 4000g
  • Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
  • Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
  • Keep on ice until use (within the next two hours).
  • Add 10μL of plasmid DNA to the 90μL of cell concentrate.
  • Keep on ice for 5 minutes.
  • Put 1mm cuvettes on ice too.
  • Pipette the cell/DNA mixture into the cuvette
  • Electrporate at 1200 volts
  • Time constant should be ~5.0
  • Immediately transfer the electroporated cells to 900μL of MRS medium.
  • Incubate at 30°C for 2-3 hours.
  • Plate on medium with the appropriate antibiotic.
  • Incubate at 30°C for two days.
  • Pick a colony