CM: Difference between revisions
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'''Instructions''' | '''Instructions''' | ||
#Make up the yeast nucleic acid hydrolysate according to the directions above: do not sterilize. Make up the vitamin solution following the recipe above and sterilize by autoclaving (115˚C, 10 min). | #Make up the yeast nucleic acid hydrolysate according to the directions above: do not sterilize. Make up the vitamin solution following the recipe above and sterilize by autoclaving (115˚C, 10 min). | ||
Dissolve all of the ingredients, except the agar and the vitamin solution, but including the glucose, in the distilled water | #Dissolve all of the ingredients, except the agar and the vitamin solution, but including the glucose, in the distilled water | ||
#Adjust the pH to 7.2 with 1N HCl | #Adjust the pH to 7.2 with 1N HCl | ||
#Add the vitamin solution | #Add the vitamin solution | ||
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==Uses== | ==Uses== | ||
*A non-selective medium useful for genetic crosses (the progeny from such crosses can subsequently be analysed on appropriate selective media such as [[MM]]<sup>[1]</sup> | |||
==Notes== | ==Notes== | ||
<sup>*</sup>The histidine, proline, tryptophan, and tyrosine should be added for the relevant auxotrophic mutants; if not working with auxotrophs, any/all of these amino acids can be omitted. | |||
==References== | ==References== | ||
Hopwood DA. Genetic analysis and genome structure in Streptomyces coelicolor. Bacteriol Rev. 1967;31(4):373–403. | [1] Hopwood DA. Genetic analysis and genome structure in Streptomyces coelicolor. Bacteriol Rev. 1967;31(4):373–403. | ||
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 | [2] 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 |
Revision as of 11:50, 31 July 2019
CM (Complete Medium)
Preparation
Per litre:
- 5g K2HPO4
- 0.5g NaCl
- 0.5g MgSO4*7H2O
- 2g Difco Bacto-peptone
- 1.5g Difco Casaminoacids
- 50mg each L-histidine, L-proline, L-tryptophan, and L-tyrosine*
- 5mL Yeast nucleic acid hydrolysate
- 1mL vitamin solution
- 25g glucose
- 10g agar
- 1L dH2O
Yeast nucleic acid hydrolysate
- Boil 2g yeast nucleic acid in 15mL 1N HCl for 10 min.
- Boil 2g yeast nucleic acid in 15mL 1N NaOH for 10 min.
- Mix the two solutions and adjust to pH6
- Filter through filter paper while hot
- Make up the volume to 40mL with water
- Do not sterilize
Vitamin solution
- 100mg riboglavine
- 100mg nicotinamide
- 10mg p-aminobenzoic acid
- 50mg pyridoxine HCl
- 50mg thiamine HCl
- 20 mg biotin
- 100mL water
Instructions
- Make up the yeast nucleic acid hydrolysate according to the directions above: do not sterilize. Make up the vitamin solution following the recipe above and sterilize by autoclaving (115˚C, 10 min).
- Dissolve all of the ingredients, except the agar and the vitamin solution, but including the glucose, in the distilled water
- Adjust the pH to 7.2 with 1N HCl
- Add the vitamin solution
- Dispense 200ml into flasks each containing 2g agar
- Autoclave
Uses
- A non-selective medium useful for genetic crosses (the progeny from such crosses can subsequently be analysed on appropriate selective media such as MM[1]
Notes
*The histidine, proline, tryptophan, and tyrosine should be added for the relevant auxotrophic mutants; if not working with auxotrophs, any/all of these amino acids can be omitted.
References
[1] Hopwood DA. Genetic analysis and genome structure in Streptomyces coelicolor. Bacteriol Rev. 1967;31(4):373–403.
[2] 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