Ferment It Yourself

Total: 20μL

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).

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

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

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.

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).

Suspend cells in 1 mL of sterile water.

Add 0.5 to 0.75 mm glass beads and vortex for 3 minutes.

Add 2.5 mL of 0.2% (wt/vol) pyrogallol dissolved in methanol and vortex for 3 minutes.

Add 1.25 mL of 60% (wt/vol) KOH and vortex for 10 seconds.

Incubate for 1 h at 75ºC, vortexing every 15 minutes.

Add appropriate amount of hexane.

Centrifuge tubes for 5 minutes at 2,800 rpm.

Pipette 1 mL of hexane into a cuvette.

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 :

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.