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
