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

• 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

Heat Shock Transformation

• Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min.
• add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C.
• put the cells back on ice for 2min.
• add 200µL of LB to the cells and incubate 2 hours at 37°C.
• plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.

Vitamin A Titration

• Take 1g of food (with particle < 1mm of diameter) in bottle protected of the light, and add 15ml of hexane. Mix it.
• Add again 15ml of hexane and shake during 5-10 minutes (do the same time, if you want compare two food sample).
• In an other bottle protected of the light, do a filtration (coarse cellulose filter) of the previous solution.
• Take the batter who don't pass the filter, and 15ml of hexane, shake 5 minutes, and filtered it like previously, to take all B-caroten as possible from the food sample.
• Put in the fridge at -20°C to conserve it.
• Obtain the result with a spectrophotometer with wavelenght of 450 nm (with a blank of hexane).

Vitamin A Titration using HPLC