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
