Difference between revisions of "Team:Virginia/Experiments"
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Revision as of 23:27, 31 October 2017
Protocols
Paracoccus denitrificans protocols
Methods:
- Grow Pc. denitrificans cultures in liquid media
- Dilute 500mL of Pc. denitrificans culture in 500mL SGM17 medium
- ***Cold Room from here on***
- Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. carefully dump out supernatant
- 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
- Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add appropriate amount of water to the other empty centrifuge bottle for counterbalance
- Centrifuge the two bottles at 5000xg at 4 C
- 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
- Centrifuge the two bottles at 5000xg at 4 C
- Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol
- Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes
- Flash freeze with liquid nitrogen and store at -80 C
