R5

R5

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
• 2 ml Trace element solution
• 5 g Difco yeast extract
• 5.73 g TES buffer
• To 1000 ml dH₂O

Pour 100 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%)
• 0.4 ml CaCl₂.2H₂O (5M, 3.68%)
• 1.5 ml L-proline (20%)
• 0.7 ml NaOH (1N) (unsterilised is okay)
• 0.75 ml Required growth factors for auxotrophs

Uses

• See R2YE - just an alternate way of making but folklore suggests less reproducible results on R5.

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