BG11 media

100XBG11:

A	B	C
Ingredient	Quantity (g/L)
NaNO3	149.6
MgSO4 7H2O	7.49
CaCl2	3.6
Citric acid	0.6
Na2EDTA	1.12 ml 0.25M solution, pH 8.0	0.61 mL 0.5 M

Trace elements

A	B	C
Ingredient	Quantity (g/L)
H3BO3	2.86
MnCL2 4H2O	1.81
ZnSO4 7H2O	0.22
Na2MoO4 2H2O	0.39
Co(NO3)2 6H2O	0.05
CuSO4 5H2O	0.08

Iron stock

A	B
Ingredient	Quantity (g/100 mL)
Ferric ammonium citrate	1.11

Phosphate stock

A	B
Ingredient	Quantity (g/100 mL)
K2HPO4	3.05

Glucose stock

A	B
Ingredient	Quantity (g/100 mL)
Glucose	9

Soidum carbonate stock

A	B
Ingredient	Quantity (g/100 mL)
Soidum carbonate	2

Autoclave all above solutions

Filter sterilize all below solutions

HEPES buffer

A	B
Ingredient	Quantity (g/100 mL)
HEPES	23.8

Adjust to pH 7.8 using NaOH

Sodium bicarb stock

A	B
Ingredient	Quantity (g/100 mL)
NaHCO3	8.4

2. Liquid BG11 growth medium

For 1 litre BG11:

10 ml 100x BG11

1 ml Trace elements

1 ml Iron stock

1 ml Na2CO3

1 ml Phosphate stock

10 ml of TES (pH 8.2)

Up to 990ml with dH2O

Autoclave

10 ml NaHCO3 stock (added after cooling).

Note: If the NaHCO3 is added before autoclaving it can cause undesired precipitation.

Note: For large scale projects 10 ml of TES can be replaced with 5 ml of HEPES to reduce the cost

Note: Mix properly before use, iron tends to precipitate.

Optional extras:

· 10 ml Glucose stock (e.g. essential for mutants without functional PSII. Final glucose concentration = 5 mM).

· Antibiotics (see point 6).

Note: If you add optional extras remember to adjust the volume of the dH2O accordingly.

3. BG11 agar plates

For 1 litre BG11 agar:

To prepare BG11 agar plates the growth medium and the agar must be autoclaved separately.

*Prepare in 1L bottle

10 ml 100x BG11

1 ml Trace elements

1 ml Iron stock

1 ml Na2CO3

1 ml Phosphate stock

10 ml of TES (pH 8.2)

3 g of sodium thiosulphate

Top up to 490ml with dH2O,

Autoclave

*Prepare in 500 ml bottle

15 g of Difco bacto-agar

Top up to 500ml with dH2O

Autoclave

Wait until both are ~60°C, mix and add:

10 ml NaHCO3 stock

Antibiotic if necessary

For 1 litre BG11 + LB agar:

To prepare BG11 + LB agar plates the growth medium and the must will be autoclaved separately.

*Prepare in 1L bottle

10 ml 100x BG11

1 ml Trace elements

1 ml Iron stock

1 ml Na2CO3

1 ml Phosphate stock

Top up to 440ml with dH2O,

Autoclave

*Prepare in 500 ml bottle

15 g of Difco bacto-agar

Top up to 500ml with dH2O

Autoclave

Wait until both are ~60°C, mix and add:

10 ml NaHCO3 stock

50 ml of sterile LB medium (premade from the media kitchen)

Note: These plates do not require the addition of 3g of thiosulphate and TES buffer since they are commonly used only for 24 hr after triparental-mating (conjugation)

For 1 litre BG11 + Sucrose agar:

To prepare BG11 + sucrose agar plates the growth medium, the agar and the sucrose will be autoclaved separately.

*Prepare in 1L bottle

10 ml 100x BG11

1 ml Trace elements

1 ml Iron stock

1 ml Na2CO3

1 ml Phosphate stock

10 ml of TES (pH 8.2)

3 g of sodium thiosulphate

Top up to 400ml with dH2O,

Autoclave

*Prepare in 500 ml bottle

15 g of Difco bacto-agar

Top up to 500ml with dH2O

Autoclave

*Prepare in 200 ml bottle

50 g of Sucrose

Top up to 100ml with dH2O

Autoclave

Wait until all are ~60°C and mix.

Note: Once you have poured the plates wrap them individually with parafilm since they are very prone to contamination.

6. Antibiotics

Add after medium is autoclaved

Chloramphenicol 25 µg/ml. Use 25 mg/ml in ethanol stock, add 1ml/L.

Spectinomycin 50 µg/ml. Use 100 mg/ml in water stock, add 0.5ml/L.

Kanamycin 50 µg/ml. Use 50 mg/ml in water stock, add 1 ml/L.

Erythromycin 50 µg/ml. Use 50 mg/ml in ethanol stock, add 1 ml/L.

Note: Antibiotic quantities above are for 1 litre of media. If your medium volume is different, add the appropriate concentration of antibiotics.