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

Yeast Lysis with NaOH

• 20 µl NaOH into PCR tubes
• Pick colonies into NaOH
• Incubate at 95°C for ~45 minutes
• Centrifugate at 8000 krpm for 10 minutes
• Use 1 µl supernatant as template in a (10 µl) PCR

Heat Shock Transformation for Yeast

• After growth, determine the titer of the yeast culture by using spectrophotometer : pipette 10µL of cells into 1mL of water in spectrophotometer cuvette and measure the OD at 600nm.
• Add 2.5x10⁸ cells to 50mL of 2X YPD in a culture flask.
• Incubate the flask in a shaking incubator at 30°C until the cell culture is at least 2x10⁷ cells.mL^-1
• Denature 1mL of carrier DNA at 99°C for 5min and chill immediately in ice.
• Harvest the yeast cells by centrifugation at 3,000g for 5min.
• Resuspend the pellet in 25mL of sterile water and centrifuge at 3,000g for 5min at 20°C. Repeat this wash with sterile water 2 times.
• Resuspend the last pellet in 1mL of sterile water.
• Transfer the cell suspension to a 1.5mL microcentrifuge tube.
• Centrifuge for 30s at 13,000g and discard the supernatant.
• Resuspend the cells in 1mL of sterile water and pipette 100µL samples into 1.5mL microcentrifuge tubes, one for each transformation.
• For each transformation :
• 240µL of PEG 3350 (50% (w/v))
• 36µL of LiAc 1.0M
• 50µL of single-stranded carrier DNA (2.0mg.mL^-1)
• 6µL of PCR product
• 28µL of water DNAse Free
• Place the tubes at 42°C for 40min.
• Centrifuge the tubes at 13,000g for 30s in a microcentrifuge tube and remove the supernatant.
• Pipette 1mL of YPD liquid medium into the transformation tube, and vortex mix to resuspend pellet.
• Incubate 3h at 30°C to ensure good antibiotic expression.
• Plate 2, 20 and 200µL of the cell suspension onto YPD medium with 200µm.mL^-1 antibiotic G418.
• Incubate the plates at 30°C for 3 days.

Ligation

• Mix the following:
  • vector 100ng
  • insert 300ng
  • 2µL T4 DNA ligase buffer 10X
  • 1µL T4 DNA ligase
  • up to 20µL of water
• incubate 10 to 15 minutes at 22°C
• incubate 5 minutes at 70°C