Jul 09, 2024
BG-11 media
- 1UIUC
Protocol Citation: is Sparrow 2024. BG-11 media. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pkzkgwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2024
Last Modified: July 09, 2024
Protocol Integer ID: 93907
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Abstract
From McCormick lab, University of Edinburgh
(written by Anja Nenninger, Grant Gale, Alejandra Schiavon and Anton Puzorjov), introduced to protocols.io by myself
Stock solutions
Stock solutions
BG11 media
100XBG11:
| 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
| Ingredient | Quantity (g/100 mL) | |
| Ferric ammonium citrate | 1.11 |
Phosphate stock
| Ingredient | Quantity (g/100 mL) | |
| K2HPO4 | 3.05 |
Glucose stock
| Ingredient | Quantity (g/100 mL) | |
| Glucose | 9 |
Soidum carbonate stock
| Ingredient | Quantity (g/100 mL) | |
| Soidum carbonate | 2 |
Autoclave all above solutions
Filter sterilize all below solutions
HEPES buffer
| Ingredient | Quantity (g/100 mL) | |
| HEPES | 23.8 |
Adjust to pH 7.8 using NaOH
Sodium bicarb stock
| Ingredient | Quantity (g/100 mL) | |
| NaHCO3 | 8.4 |
Liquid BG11
Liquid BG11
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.
BG11 agar
BG11 agar
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
BG11 LB agar
BG11 LB agar
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)
BG11 Sucrose
BG11 Sucrose
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.
Antibiotics
Antibiotics
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.