TITLE: BG-11 media
AUTHORS: is Sparrow

[DESCRIPTION]
From McCormick lab, University of Edinburgh
(written by Anja Nenninger, Grant Gale, Alejandra Schiavon and Anton Puzorjov), introduced to protocols.io by myself



[STEPS]
SECTION: Stock solutions
1. BG11 media
 
100XBG11:

 

 
 
Trace elements
 

 

 
Iron stock
 

 

 
Phosphate stock
 
 

 

Glucose stock
 
 

 

Soidum carbonate stock
 
 

 

 
Autoclave all above solutions
 
 
 
Filter sterilize all below solutions
HEPES buffer
 
 

 

Adjust to pH 7.8 using NaOH
 


Sodium bicarb stock
 

 

 
 
 
 

 
 


SECTION: Liquid BG11
2. 
 
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.
 
SECTION: BG11 agar
3. 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
 

 
SECTION: BG11 LB agar
4. 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)
SECTION: BG11 Sucrose
5. 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.
SECTION: Antibiotics
6.  
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.