MaintenanceofProbesinbacteriaincludingEscherichiacoli

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.