Team:Virginia/Experiments



Protocols


Paracoccus denitrificans protocols

Methods:

  1. Grow Pc. denitrificans cultures in liquid media
  2. Dilute 500mL of Pc. denitrificans culture in 500mL SGM17 medium
  3. ***Cold Room from here on***
  4. Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. carefully dump out supernatant
  5. Completely resuspend(shake in side to side motion to avoid liquid loss) the pellets from the previous step in all centrifuge bottles each with 50mL of 0.5 M sucrose solution with 10% glycerol
  6. Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add appropriate amount of water to the other empty centrifuge bottle for counterbalance
  7. Centrifuge the two bottles at 5000xg at 4 C
  8. 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
  9. Centrifuge the two bottles at 5000xg at 4 C
  10. Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol
  11. Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes
  12. Flash freeze with liquid nitrogen and store at -80 C