Nalidixic Acid → Nalidixic Acid

Participants

Reaction Steps

The culture medium LB (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) that once autoclaved was supplemented with 25 μg/mL of kanamycin was used to grow E. coli S17-1 λ pir. Once the stationary phase was achieved, 0.2-0.3 A600 units of the App culture and 0.6-0.8 A600 units of the E. coli culture were added to 1 mL of a 10 mM solution of MgSO4. Next it was centrifuged during 2 minutes at 15,000 g and the pellet so obtained was resuspended in 200 μl of a 10 mM MgSO4 solution. Once the mixture of both cultures had been done, this was extended on a 2.5 cm and 0.45 μM nitrocellulose filter previously placed on a Petri dish containing TSYN medium supplemented with 15 g/L Noble agar. After incubation during 6 hours at 37 C., the filter with the conjugation was placed in a tube containing 2 mL of PBS (Na2HPO410 mM, KH2PO4 1 mM, NaCl 137 mM, KCl 2 mM pH 7.4). After vigorous shaking, the filter was removed and the cell suspension was centrifuged during 2 minutes at 15,000 g and the pellet was resuspended in 500 μL of PBS. The so obtained suspension was distributed in Petri dishes with TSYN medium supplemented with 15 g/L Noble Agar, 50 μg/mL kanamycin and 50 μg/mL nalidixic acid, at a rate of 100 μL of cell suspension for each Petri dish. The resulting cultures were incubated at 37 C. for 24-36 hours. With this procedure 65 colonies resistant to kanamicin and nalidixic acid, were obtained for the conjugation with the plasmid pApxIΔH2, which equals a frequency of transformation of 1.3×10−7 for each receptor cell.

Provenance & Evidence