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

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</div>
 
</div>
  
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<div class="textBox" id="HeatShockYeast">
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  <h3>Heat Shock Transformation for Yeast</h3>
 +
<ul>
 +
<li>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.</li>
 +
<li>Add 2.5x10<sup>8</sup> cells to 50mL of 2X YPD in a culture flask.</li>
 +
<li>Incubate the flask in a shaking incubator at 30°C until the cell culture is at least 2x10<sup>7</sup> cells.mL<sup>-1</sup></li>
 +
<li>Denature 1mL of carrier DNA at 99°C for 5min and chill immediately in ice.</li>
 +
<li>Harvest the yeast cells by centrifugation at 3,000g for 5min.</li>
 +
<li>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.</li>
 +
<li>Resuspend the last pellet in 1mL of sterile water.</li>
 +
<li>Transfer the cell suspension to a 1.5mL microcentrifuge tube.</li>
 +
<li>Centrifuge for 30s at 13,000g and discard the supernatant.</li>
 +
<li>Resuspend the cells in 1mL of sterile water and pipette 100µL samples into 1.5mL microcentrifuge tubes, one for each transformation.</li>
 +
<li>For each transformation :</li>
 +
<ul>
 +
    <li>240µL of PEG 3350 (50% (w/v))</li>
 +
    <li>36µL of LiAc 1.0M</li>
 +
    <li>50µL of single-stranded carrier DNA (2.0mg.mL<sup>-1</sup>)</li>
 +
    <li>6µL of PCR product </li>
 +
    <li>28µL of water DNAse Free</li>
 +
</ul>
 +
<li>Place the tubes at 42°C for 40min.</li>
 +
<li>Centrifuge the tubes at 13,000g for 30s in a microcentrifuge tube and remove the supernatant.</li>
 +
<li>Pipette 1mL of YPD liquid medium into the transformation tube, and vortex mix to resuspend pellet.</li>
 +
<li>Incubate 3h at 30°C to ensure good antibiotic expression.</li>
 +
<li>Plate 2, 20 and 200µL of the cell suspension onto YPD medium with 200µm.mL<sup>-1</sup> antibiotic G418.</li>
 +
<li>Incubate the plates at 30°C for 3 days.</li>
 +
</ul>
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</div>
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<div class="textBox" id="Ligation">
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<h3>Ligation</h3>
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<ul>
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<li>Mix the following:
 +
 +
      <ul>
 +
        <li>vector 100ng</li>
 +
        <li>insert 300ng</li>
 +
        <li>2µL T4 DNA ligase buffer 10X</li>
 +
        <li>1µL T4 DNA ligase</li>
 +
        <li>up to 20µL of water</li>
 +
      </ul></li>
 +
      <li>incubate 10 to 15 minutes at 22°C</li>
 +
      <li>incubate 5 minutes at 70°C</li>
 +
</ul>
 +
</div>
  
  
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     <div class="innerBox yellow">
 
     <div class="innerBox yellow">
       <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Heat shock transformation for yeast">
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       <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#HeatShockYeast">
 
         <div class="ptext"><p>Heat shock transformation for yeast</p></div>
 
         <div class="ptext"><p>Heat shock transformation for yeast</p></div>
 
         <img src="https://static.igem.org/mediawiki/2015/e/ec/ParisBettencourt_protocolsHeatBlock.JPG">
 
         <img src="https://static.igem.org/mediawiki/2015/e/ec/ParisBettencourt_protocolsHeatBlock.JPG">

Revision as of 09:52, 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

  • 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.
  • 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