TITLE: Recipe for standard BG-11 media
AUTHORS: Anna  Behle, Alice   Pawlowski

[DESCRIPTION]
Stanier RY, Deruelles J, Rippka R, Herdman M, Waterbury JB: Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 1979, 111:1–61.
Recipes for standard and alternative BG11 for culturing freshwater cyanobacteria, such as Synechocystis sp. PCC 6803, as described.
Media is usually not suitable for marine cyanobacteria.
 Final Concentration of Medium.
CaCl2*2 H2O                        0.036 g/L
Citric acid                              0.006 g/L
NaNO3                                   1.4958 g/L
MgSO4* 7 H2O                     0.0749 g/L
0.25M Na2EDTA (pH 8)      0.0056 mL/L
Na2CO3                                 20 µg/ml
Fe(III) Ammonium citrate   6 µg/ml
K2HPO4 * 3H2O                   30 µg/ml
TES Buffer (pH 8)                10 mM
H3BO3                                   2.86 mg/L
MnCl2 * 4 H2O                     1.81 mg/L
ZnSO4 * 7 H2O                      0.222 mg/L
Na2MoO4 * 2 H2O                0.390 mg/L
Co(NO3)2 *6 H2O                0.049 mg/L
(CuSO4 * 5 H2O                    0.079 mg/L if required)    

[BEFORE_START]
For plates:
Thaw antibiotic stocks before pouring plates.

[GUIDELINES]
Always work under sterile conditions when handling sterile media or stocks. Work under the clean bench.

[STEPS]
SECTION: 100 x BG11 stock:
1. CaCl2 2H2O (3.6 g · L-1)
Citric acid (0.6 g · L-1)
NaNO3 (149.58 g · L-1)
MgSO4 · 7 H2O (7.49 g · L-1)
0.25 M Na2-EDTA, pH 8.0  (0.56 ml · L-1)
 

SECTION: Supplemental stocks for standard media:
2. 1000x Na2CO3: 20 mg mL-1
100x TES-buffer, pH 8.0 (1M), adjust with KOH
1000x K2HPO4 x 3 H2O: 30 mg · mL-1
1000x Fe(III) ammonium citrate (6 mg ·m L-1)
5000x CuSO4 5 H2O (395 ng · mL-1) (sterilize using a filter)
SECTION: Trace metal mix:
3. 1000x concentration:
H3BO3 (2.86 g · L-1)
MnCl2 · 4 H2O (1.81 g · L-1)
ZnSO4 · 7 H2O (0.222 g · L-1)
Na2MoO4 · 2 H2O (0.390 g · L-1)
Co(NO3)2 · 6 H2O (0.049 g · L-1)
 
For BG11 lacking certain metals (e.g. for working with metal inducible promoters PpetE, PcoaT, PziaA etc., trace metal mix can be prepared lacking these chemicals and used instead of standard trace metal mix.
SECTION: Standard 1x BG11
4. Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.
 
 
   Stock solution Volume   100x BG11 Stock 10 mL   1000x Na2CO3 1 mL   1000x K2HPO4 x 3 H2O 1 mL   100x TES-buffer 10 mL   1000x Trace Metal Mix 1 mL   
 
Add ultra pure water to 1 L.
Autoclave.
After autoclaving, add 1 mL 1000x Fe(III) ammonium citrate.
Optional: After autoclaving, add 200 µL 5000x CuSO4
 
SECTION: Standard 1x BG11 -N
5. Fill 1 L bottle with 500 mL ultra pure water. Add stock solutions as shown below.
 
 
   Stock solution Volume   100x BG11 Stock -N 10 mL   1000x Na2CO3 1 mL   1000x K2HPO4 x 3 H2O 1 mL   100x TES-buffer 10 mL   1000x Trace Metal Mix 1 mL   
 
Add ultra pure water to 1 L.
Autoclave.
After autoclaving, add 1 mL sterile 1000x Fe(III) ammonium citrate.
Optional: After autoclaving, add 200 µL sterile 5000x CuSO4
SECTION: Standard 2x BG11 for agar plates
6. Fill 500 mL bottle with 250 mL ultra pure water. Add stock solutions as shown below.
 
 
   Stock solution Volume   100x BG11 Stock -N 10 mL   1000x Na2CO3 1 mL   1000x K2HPO4 x 3 H2O 1 mL   100x TES-buffer, pH = 8.0 10 mL   1000x Trace Metal Mix 1 mL   
 
Add ultra pure water to 500 mL.
Autoclave.
After autoclaving, add 1 mL sterile 1000x Fe(III) ammonium citrate.
Optional: After autoclaving, add 200 µL sterile 5000x CuSO4
SECTION: BG11 plates
7. Prepare 1.5 % agar: Weigh 4.5 g Bacto Agar. Fill up to 300 mL. Autoclave.
 
Microwave agar until liquid. Let cool.
 
 
8. In a 50 mL Falcon, add 1 vol 2x BG11 and 1 vol liquid 1.5 % agar. (Note: Usually, one plate requires 30-40 mL total volume.)
9. When mixture is hand warm, add appropriate antibiotics, if required. Quickly pour plate, avoiding air bubbles.
SECTION: 100 x BG11 stock:
1.1. For BG11-N prepare a 50 X stock solution without NaNO3:

CaCl2 x 2 H2O: 1.8 g/L
Citric acid: 0.3 g/L
MgSO4 x 7 H2O: 3.75 g/L
0.25 M Na2-EDTA, pH8.0: 0.28 ml/L