Cyanobacteria cultivation

BG-11 medium for cyanobacterial cultivation (for Synechocystis  and Synechococcus species)

1. Making Six stock solutions

Stock Sol. I

Citric acid 0.6 g 

Ferric ammonium citrate 0.6 g
Na2EDTA 0.1 g ／200 ml 

Stock Sol. II

NaNO3 30 g
K2HPO4 0.78 g 
MgSO4・7H2O 1.5 g ／１L 

Stock Sol. III

CaCl2・2H2O 3.8 g ／200 ml 

Stock Sol. IV

Na2CO3 4 g／200 ml 

Stock Sol. V

H3BO3 2.86 g
MnCl2・4H2O 1.81 g
ZnSO4・7H2O 0.22 g
CuSO4・5H2O 0.08 g
Na2MoO4・2H2O 0.021 g
H2SO4 50 uL 10% H2SO4 solutoin
Add MilliQ to 1L

mix them and then add cobalt powder
Co(NO3)2・6H2O 0.0494 g 

1 M HEPES-KOH (pH7.8)

dissolve HEPES power and adjust the pH to 7.8 by KOH.

Stock Sol. I is sterilized by filtration and 1 M HEPES-KOH is sterilized by autoclave.

All stock solutions are stored at 4℃

2. Making BG-11 medium
Add 
50 mL Stock Sol. II, 
20 mL HEPES-KOH, 
2 mL Stock Sol. III, 
1 mL Stock Sol. IV
1 mL Stock Sol. V. to 900 mL MillQ 
and then add MilliQ to 1L.
After autoclave, add 2 mL Stock Sol. I to 1 L BG-11 medium. BG-11 medium is stored at room temperature.

3. Cultivation conditions
i. Light
White Light, 30~100 μmol photons m-2s-1, at 25~34℃.
（In our case, 50~70 μmol photons m-2s-1, 30℃）
ii. Air
Bubbling 1~3% CO2 to liquid BG-11 medium during cultivation
iii. Growth monitoring
The growth can be monitored by measuring OD730 or OD750.

4. Making freeze stocks
Concentrate 20~30 mL cells (OD730 = 1~2) to 500 μL by centrifugation (at Room temperature)
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Add 40 μL DMSO and mix thoroughly
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Store at -80℃