R2: Difference between revisions
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*0.5 ml NaOH (1N) (unsterilised is okay) | *0.5 ml NaOH (1N) (unsterilised is okay) | ||
*0.75 ml Required growth factors for auxotrophs | *0.75 ml Required growth factors for auxotrophs | ||
Revision as of 13:21, 4 September 2019
R2
Preparation
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:
- 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
Uses
- Protoplasts regeneration
Notes
Streptomyces clavuligerus does not utilise glucose and it must be substituted by glycerol.
References
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
Hopwood DA, Wright HM (1978) Bacterial protoplast fusion-. recombination in fused protoplasts of Streptomyces coelicolor. Mol.Gen.Genet., 162, 307-317
Okanishi M, Suzuki K, Umezawa H (1974) Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. J.Gen.Microbiol., 80 389-400