Difference between revisions of "Team:Virginia/Experiments"

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<li>1. Grow Paracoccus cultures in liquid media
 
<li>1. Grow Paracoccus cultures in liquid media
 
<li>2. Dilute 500mL of Paracoccus denitrificans culture in 500mL SGM17 medium  
 
<li>2. Dilute 500mL of Paracoccus denitrificans culture in 500mL SGM17 medium  
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<br><li>***COLD ROOM FROM HERE***<br>
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<br><br><li>***COLD ROOM FROM HERE***<br><br>
 
<li>3.Harvest by centrifuge 250mL samples each in 2 four centrifuge bottles at 5000xg at 4 C. Carefully dump out supernatant.
 
<li>3.Harvest by centrifuge 250mL samples each in 2 four centrifuge bottles at 5000xg at 4 C. Carefully dump out supernatant.
 
<li>4.Completely resuspend (shake vigorously without up & down motion to avoid liquid loss) the pellets from the previous step in all centrifuge bottles each with 10050mL of 0.5 M sucrose solution with 10% glycerol.
 
<li>4.Completely resuspend (shake vigorously without up & down motion to avoid liquid loss) the pellets from the previous step in all centrifuge bottles each with 10050mL of 0.5 M sucrose solution with 10% glycerol.
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<button data-toggle="collapse" data-target="#pdtransformation">Transformation via Electroporation</button>
 
<button data-toggle="collapse" data-target="#pdtransformation">Transformation via Electroporation</button>
  
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<div id="pdtransformation" class="collapse"><br>
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textextextextextextextext
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<li>1. Thaw and mix 50 uL portions cells with 5 uL DNA
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<li>2. Transfer to ice cold electroporation cuvette
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<li>3. Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
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<li>4. The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
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<li>5. IMMEDIATELY following electroporation: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
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<li>6. Dilutions were made in the SGM17 media
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<li>7. Incubate cells at 30 C for 2 hours
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<li>8. Take 100 uL portions and plate.
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Revision as of 20:05, 31 October 2017


Paracoccus denitrificans protocols


  • 1. Grow Paracoccus cultures in liquid media
  • 2. Dilute 500mL of Paracoccus denitrificans culture in 500mL SGM17 medium

  • ***COLD ROOM FROM HERE***

  • 3.Harvest by centrifuge 250mL samples each in 2 four centrifuge bottles at 5000xg at 4 C. Carefully dump out supernatant.
  • 4.Completely resuspend (shake vigorously without up & down motion to avoid liquid loss) the pellets from the previous step in all centrifuge bottles each with 10050mL of 0.5 M sucrose solution with 10% glycerol.
  • 5. Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add 200mL water to the other empty centrifuge bottle for counterbalance.
  • 6. Centrifuge the two bottles at 5000xg at 4 C.
  • 7. Dump out supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water.
  • 8. Centrifuge the two bottles at 5000xg at 4 C.
  • 9. Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol.
  • 10. Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes.
  • 11. Flash freeze with liquid nitrogen and store at -80 C.


  • 1. Thaw and mix 50 uL portions cells with 5 uL DNA
  • 2. Transfer to ice cold electroporation cuvette
  • 3. Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
  • 4. The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
  • 5. IMMEDIATELY following electroporation: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
  • 6. Dilutions were made in the SGM17 media
  • 7. Incubate cells at 30 C for 2 hours
  • 8. Take 100 uL portions and plate.

    • Measurement protocols

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