Team:Paris Bettencourt/Protocols

PCR protocol

PCR protocol (Phusion)
  • Prepare the following mix
    • 2µL primer Forward (10 µM)
    • 2µL primer Reverse (10 µM)
    • 0.1 to 1 ng of template DNA
    • 25µL of Life Technologies Phusion High-Fidelity PCR Master Mix with HF Buffer
    • complete to 50µL of DNase/RNase-free water
  • Launch 30 to 35 PCR cycles using the following parameters:
    time (min) temperature (°C) function
    3:00 98 melting
    0:30 98 melting
    0:30 XX°C annealing
    1 minute/kb 72 extension
    10:00 72 extension
    infinite hold 12 storage

Gel purification with the QIAquick® Gel Extraction Kit

Gel-Purification protocol with the QIAquick® Gel Extraction Kit
  • Excise the DNA band from the gel as precisely as possible and put it in a micro-centrifuge tube.
  • Add 3 volume of QG buffer (100mg of gel ~ 100ul) in the tube and incubate for 10 min at 50°C (or 10 min on a vortex).
  • Add one volume of isopropanol and mix.
  • Centrifugate the tube for 2 min at 14 000 rpm.
  • Carefully pipette the supernanant in a QIAquick column.
  • Put the column in a 2ml microcentrifuge tube and centrifuge it for 1 min at 14 000 rpm.
  • Discard the flow-through.
  • Add 750 uL of PE buffer in the column and centrifuge for 1 min and discard flow-through.
  • Centrifuge again for 2 min and put the column in a new 1.5uL microcentrifuge tube.
  • Elute the DNA by putting 50uL of water in the middle of the column and let it stand for 2 min.
  • Centrifugate for 2min at 10 000 rpm

Gel Electrophoresis with SYBR safe

Gel-Electrophoresis protocol
  • Preparation of the Gel (50mL)
    • Mix 0.5 grams of agarose with 50mL 0.5% TAE solution
    • Heat the Erlenmeyer (conical flask) in a microwave until the liquid becomes totally transparent.
    • Let it rest until you can take the Erlenmeyer in your hand without pain for 10 seconds.
    • Add 5uL of SYBR® Safe (10000X) and mix gently until the solution homogenizes.
    • Pour the liquid in a gel mold with the desired number of wells and wait 20-30 min (or until the gel is solidified).
    • Put the gel inside the electrophoresis machine and pour TAE 0.5% until the gel is totally submerged.
  • Preparation of the samples
    • Mix 5uL of each sample with 1uL of 6X loading dye in microcentrifuge tubes (or any hydrophobic surface like parafilm)
    • Pipette 5-6uL of each sample inside a different well.
    • Pipette 5uL of the appropriate ladder in one of the wells (Some ladders have a dye in it, but some don't, so one has to add gel loading dye to these ones)
    • Close the machine and launch the migration with the desired voltage
    • After the desired migration time turn off the machine, remove the lid and put the gel inside the gel visualizer.

Titration of phytic acid

Titration of phytic acid using Megazyme© Kit
  • Preparation of reagent solutions (not supllied) :
    • Trichloroacetic acid (50% w/v) : 100mL
      • Add 50g of thrichloroacetic to 60mL of distilled water and dissolve. Make to volume (100mL) with distilled water. (Stable for > 6 moths at 4°C)
    • Hydrochloric acid (0.66M) : 1L
      • Add 54.5mL of hydrochloric acid to 945.5mL of distilled wtaer an mix. (Store at room temperature)
    • Sodium hydroxide (0.75M) : 200mL
      • Add 6g of sodium hydroxide pellets to 180mL of distilled water and dissolve. Make to volume (200mL) with distilled water. (Store at room temperature)
    • Phytic acid :
      • Pure phytic acid control sample may be required.
  • Sample extraction :
    • Weigh 1g of sample material.
    • Add 20mL of hydrochloric acid (0.66M).
    • Cover with foil and incubate for a minimum of 3 hours at room temperature.
    • Transfer 1mL of extract to a 1.5mL microcentrifuge tube.
    • Centrifuge 10 minutes at 13,000rpm.
    • Transfer 0.5mL of the resulting extract supernatant to a fresh 1.5mL microfuge tube.
    • Add 0.5mL of sodium hydroxide solution (0.75M) to neutralise.
  • Enzymatic dephosphorylation reaction :
  • Colourimetric determination of phosphorous :
  • Preparation of phosphorous calibration curve :
  • Calculation :
    • Phosphorous calibration curve :
      • Determine the absorbance of each phosphorous standard. Substract the absorbance of STD0 from the absorbance of the others STD, therby obtaining ΔA(phosphorous). Calculate M as follows, for each standard: \[ \begin{align} M = \frac{P(\mu g)}{\Delta A (phosphorous)} \end{align} \] Use "Mean M" to calculate the phosphorous content of test samples.
    • Phosphorous / phytic acid content :
      • Determine the absorbance of the both "Free Phosphorous" and "Total Phosphorous". Substract the absorbance of the "Free Phosphorous" sample from the absorbance of the "Total Phosphorous" sample. Obtaining ΔA(phosphorous). \[ \begin{align} C^{o} = \frac{mean(M)\times 20 \times F}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous) \end{align} \]
      Where :
      \(20\) = original sample extract volume
      \(F\) = dilution factor
      \(10000\) = conversion from µg/g to g/100g
      \(1.0\) = wigh of original sample material
      \(\nu\) = sample volume

      It follows for phosphorous :
      \[ \begin{align} C^{o} ~= \frac{mean(M)\times 20 \times 55.6}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)\\ ~= mean(M) \times 0.1112 \times \Delta A(phosphorous) \end{align} \] It follows for phytic acid :
      \[ \begin{align} C^{o} = \frac{phosphorus}{0.282} \end{align} \]

Vitamin B12 titration

  • Disrupt the microorganisms in a 0.1 N phosphate buffer solution containing 0.01% KCN at pH 5.5 for 20 min at 100°C.
  • Vitamin B12 formed intracellularly is determined spectrophotometrically in the dicyano-form at wavelength of 367 nm.

    • PCR purification

    • Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down
    • Transfer in a centrifugation column
    • Centrifuge 1 min at 14000 rpm
    • Throw the filtration product
    • Add 700μL of washing solution
    • Centrifuge 1 min at 14000 rpm
    • Throw the filtration product
    • Add 500μL of washing solution
    • Centrifuge 1 min at 14000 rpm
    • Throw the filtration product
    • Centrifuge 1 min at 14000 rpm
    • Throw the filtration product
    • Put the column in a sterile 1.5mL microcentrifuge tube
    • Add 45μL of DNAse/RNAse free water on the membrane
    • Wait 2 minutes
    • Centrifuge 2min at 10000rpm
    • Discard the column, DNA is saved in water

    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).

    BANANAAAA

    • Line up ice cream scoops next to each other in an oval deep dish or a banana boat.
    • Cut the ends of the banana off (about 1/4 inch) while still in the peel.
    • Slice in half longways.
    • Pop each half of the banana out of the peel onto each side of the ice cream row, pressing down and in a little so it'll stay put.
    • Top the vanilla ice cream with the pineapple, the chocolate with the chocolate syrup and the strawberry with the strawberry sauce.
    • Spoon the wet walnuts over all three scoops of ice cream.
    • Top each scoop with some whipped cream and a cherry for each.
    • Enjoy!

    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 a 250mL LB flasks with 2.5mL of an overnight culture of cells
    • Incubate until the the DO600 reach 0.6 to 0.8
    • Place the cultures on ice for 15 minutes
    • Pour the culture in cold sterile 50mL falcon tubes
    • Centrifuge for 10 minutes at 4000rpm, 4°C
    • Throw the supernatant
    • Resuspend the cells in 50mL cold distilled water
    • Place the cells on ice for 10 minutes
    • Centrifuge them for 10 minutes at 4000rpm, 4°C
    • Throw the supernatant
    • Resuspend the cells in 12.5mL cold 10% glycerol
    • Place the cells on ice for 10 minutes
    • Centrifuge them for 10 minutes at 4000rpm, 4°C
    • Throw the supernatant
    • Resuspend the cells in 5mL cold 10% glycerol
    • Make aliquots of 50 to 100µL 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

    • 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.

    Lactococcus lactiselectrocompetent preparation

    Prepare the following solutions
    • Glycine/Sucrose solution
      • 80mL of 50% sucrose solution
      • 29.2mL of 20% glycine solution
      • 7.6mL of water
    • Electroporation buffer
      • 0.125g of NaHPO4
      • 34.2mL of 50% sucrose solution
      • 100µL of MgCl2 1M
      • 65.5mL of water
    • Electroporation buffer + glycreol
      • 187.5µL of 80% glycerol
      • 812.5µL of electroporation buffer
    Protocol:
    • Inoculate 10mL of M17 + 1% glucose (GM17) with some Lactococcus and incubate overnight at 30°C
    • Inoculate a 100mL flask of GM17 with the 10mL of overnight culture
    • Incubate the flask at 30°C until DO reach 0.4 to 0.5
    • Add 100mL of the glycine/sucrose solution
    • Incubate 1 hour at 30°C with shaking
    • Split the 100mL culture in twyo 50mL centrifuge tubes
    • Centrifuge the cells 10 minutes at 6000 rpm
    • Throw the supernatant and work on ice for the rest of the protocol
    • Resuspend the cells in 20mL of electroporation buffer
    • Centrifuge the cells 10 minutes at 6000 rpm and then throw the supernatant
    • Resuspend the cells in 20mL of electroporation buffer
    • Centrifuge the cells 10 minutes at 6000 rpm and then throw the supernatant
    • Resuspend the cells in 0.5mL of electroporation buffer
    • Centrifuge the cells 10 minutes at 6000 rpm and then throw the supernatant
    • Resuspend the cells in 1mL of cold electroporation buffer + glycerol
    • Aliquots the cells in eppendorf tubes
    • Save the cells at -80°C

    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

    Vitamin B2 Titration using HPLC

    Idli Recipe

    • Soak rice and dall separately for 4-5 hours. (for 1.5 volume of rice, put the same volume of water, and 0.5 volume of dall, put the same volume of water).
    • Rinse both, mix it, add 2/3 volume of water of this volume.
    • Blend this mix, and let ferment for 12-16 hours.
    • Then Cook it with idli cooker for 15 minutes.
    In the case where we add micro-organisms, we have 3 options:
    1. Add µorganisms during the soak phase
    2. Add µorganisms after the blending phase.
    3. Let soak µorganisms in the same time that rices and dall, and add it for the blending phase.
    The third was mainly used.

    Miniprep protocol

    • centrifuge an overnight culture of cells 10min at 4krpm
    • throw the filtrate
    • resuspend the pellet in 250μL of Cell Resuspension Solution then mix it
    • transfer it in a 1.5mL microcentrifuge tube
    • add 250mL of Cell lysis solution and mix by inverting several times
    • incube until the liquid is clear, maximum 5min
    • add 10μL of Alkalyne protease solution, mix by inverting several times and incube 3 to 4 min
    • add 350μL of Neutralisation Solution and mix by inverting several times
    • centrifuge 10min at 14krpm
    • add the supernatant in a column
    • centrifuge 1min, 14krpm, then throw the filtrat
    • add 750μL Washing Solution
    • centrifuge 1min, 14krpm, then throw the filtrat
    • add 250μL Washing Solution
    • centrifuge 2min, 14krpm, then throw the filtrat
    • centrifuge 1min, 14krpm
    • transfer the column in a sterile microcentrifuge 1.5ml tube
    • add 50μL of DNAse/RNAse free water right on the membrane of the filter, wait 1min
    • centrifuge 1min, 14krpm
    • throw the column, plasmid is saved in water


    Miscellaneous