Team:Virginia/Experiments



Protocols


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


    Isolate Single colonies
  • 1. Select glycerol stock cultures from -80 C storage to begin
  • 2. Thaw on ice for 15 minutes and observe when the cell stock begins to melt
  • 3. Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
  • If the culture is P. denitrificans, culture on PD media or standard LB
  • If the culture is E. coli use LB media
  • If using untransformed bacteria, equivalent non-selective media should be used
  • 4. Incubate both P. denitrificans and E. coli overnight at 37 C
  • 5. Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics

    Preculture for Device Testing

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