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Maintenance of Probes in bacteria including <I>Escherichia coli</I>

Maintenance of Probes in bacteria including Escherichia coli

Plasmid (pUC series) containing genomic DNA fragments are maintained in E. coli strain DH5aTM. The E. coli cultures are routinely cultured at 37 C on Luria-Bertani (LB) agar on or in LB broth containing Ampicillin (30 µg/ml) or Carbenicillin (50 µg/ml broth, 100 µg/ml agar). E. coli strains are usually preserved in stab agar or glycerol for mid-term storage and lyophilized for long-term storage.

Storage in Stab Agar: Most strains of E. coli can be stored for one to two years in stab agar.

  1. Use airtight, autoclaved vials containing 2/3 full (appr. 1 ml) of medium.

  2. Inoculate each vial with a single colony.

  3. Leave the cap of the vial sightly loose and incubate overnight at 37 C.

  4. Seal the vial tightly and stored at room temperature in a dark place.

  5. To revive a stored strain, streak onto LB plate containing appropriate antibiotics for a single colony.

Storage in glycerol Solution: Most strains of bacteria including E. coli,and fungi (spores is preferred) can be stored for one to two years in glycerol solution at -20 C. At -70 C they can be stored almost for life time.

  1. Prepare 0.5 ml of glycerol solution in 2.0 ml screw-cap vials, autoclave (15 min, 121 C), cool, and store them at room temperature.

  2. Transfer one drop (0.5 ml) of an overnight-grown culture into a vial.

  3. Voltex, store at -20 or -70 C (-70 C storage is preferred for long-term maintenance).

  4. To revive a stored strain, streak onto LB plate containing appropriate antibiotics for a single colony.