R2YE medium is a variation of R2 containing yeast extract.
Make up the following solution:
- 103 g Sucrose
- 0.25 g K2SO4
- 10.12 g MgCl2.6H2O
- 10 g Glucose
- 0.1 g Difco Casaminoacids
- 800 ml dH2O
Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar. Sterilise by autoclaving. At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:
- 5 ml Difco yeast extract (10%)
- 1 ml KH2PO4 (0.5%)
- 8 ml CaCl2.2H2O (3.68%)
- 1.5 ml L-proline (20%)
- 10 ml TES buffer (5.73%, adjusted to pH7.2)
- 0.2 ml Trace element solution
- 0.5 ml NaOH (1N) (unsterilised is okay)
- 0.75 ml Required growth factors for auxotrophs
- Protoplasts regeneration
Streptomyces clavuligerus does not utilise glucose and it must be substituted by glycerol.
Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK
Thompson CJ, Ward JM, Hopwood DA (1980) DNA cloning in Streptomyces: resistance genes from antibiotic-producing species. Nature, 286, 525-527.