TITLE: BG11_and_inducer_preparation
AUTHORS: maurice.mager1808

[DESCRIPTION]
Protocol for the preparation of media and inducers used during the interlaboratory study published by Mager et al. 2023.

[STEPS]
SECTION: HEPES buffer preparation
2. 
  Buffer solutiong/LHEPES, NaOH (pH 8)240

SECTION: Stock solution 1 preparation
3. 
 
  Stock 1g/LNaNO3600CaCl2 * 2H2O14.4

SECTION: Stock solution 2 preparation
4. 
  Stock 2g/LMgSO4 * 7H2O30FeCl3 * 6H2O1.62Na2EDTA * 2 H2O2.24Micronutrients400 mL

SECTION: Stock solution 3 preparation
5. 
  Stock 3g/LKH2PO416Na2EDTA * 2 H2O5.2
 
SECTION: Stock solution 4 preparation
6. 
  Stock 4g/LNaHCO380

SECTION: Micronutrient stock solution preparation
7. 
  Micronutrientsg/LH3BO32.86MnCl2 * 4H2O1.81ZnSO4 * 7H2O0.222Na2MoO4 * 2H2O0.391CuSO4 * 5H2O0.079*Co(NO3)2 6H2O0.049

*  Copper sulfate is not to be added for the interlab study as we need copper free media for PpetE induction
SECTION: Rhamnose stock preparation
8. 

 


SECTION: Copper sulfate stock preparation
9.  

SECTION: HEPES buffer preparation
2.1. Dissolve 24g of HEPES in 40 ml of MilliQ water
SECTION: HEPES buffer preparation
2.2. Under stirring adjust pH to 8 using NaOH
SECTION: HEPES buffer preparation
2.4. Filter sterilize the solution
SECTION: Stock solution 1 preparation
3.1. Dissolve 1.4g CaCl2*2H2O in 70 ml of MilliQ water
SECTION: Stock solution 1 preparation
3.2. Add 60g of NaNO3 under vigorous stirring at 50°C until fully dissolved.
SECTION: Stock solution 1 preparation
3.3. Cool solution to room temperature and add MilliQ water to 100 ml.
SECTION: BG11-PC overview
1. BG11 medium for interlab study (after van Alphen et al., 2018)

 
 
   MacronutrientsmMg/LNaNO317.651.5KH2PO40.2940.04MgSO4 * 7H2O0.3040.075CaCl2 * 2H2O0.2440.036FeCl3 * 6H2O0.0150.004Na2EDTA * 2 H2O0.050.0186NaHCO39.441HEPES51.2MicronutrientsµMmg/LH3BO346.252.86MnCl2 * 4H2O9.151.81ZnSO4 * 7H2O0.770.222Na2MoO4 * 2H2O1.620.391CuSO4 * 5H2O0.3160.079Co(NO3)2 6H2O0.1680.049

 
 

 
SECTION: BG11-PC overview
1.1. 
  ComponentmL/LBuffer solution5 mLStock 12.5 mLStock 22.5 mLStock 32.5 mLStock 410.5 mLFill with autoclaved water to 1L


 
SECTION: HEPES buffer preparation
2.3. Add MilliQ water to 100 ml
SECTION: Stock solution 1 preparation
3.4. Filter sterilize the solution


SECTION: Stock solution 2 preparation
4.1. Dissolve 0.224g EDTA Na2*2H2O  in 40 mL MilliQ water followed by 0.162g FeCl3·6H2O and leave the solution stirring over night
SECTION: Stock solution 2 preparation
4.2. Dissolve 3g MgSo4*7H2O in 15 ml MilliQ water and add it to the EDTA Na2*2H2O + FeCl3·6H2O solution
SECTION: Stock solution 2 preparation
4.3. Add 40 ml of Micronutrient solution (See Section Micronutrient stock solution preparation) to this mix and fill to 100 ml using MilliQ water
SECTION: Stock solution 2 preparation
4.4. Filter sterilize the solution.
SECTION: Stock solution 3 preparation
5.1. Dissolve 1.6 g K2HPO4 in 40 ml of MilliQ water
SECTION: Stock solution 3 preparation
5.2. Dissolve 0.52 g EDTA Na2*2H2O in the same solution
SECTION: Stock solution 3 preparation
5.4. Filter sterilize the solution
SECTION: Stock solution 3 preparation
5.3. Fill to 100 ml using MilliQ water

SECTION: Stock solution 4 preparation
6.1. Dissolve 8 g NaHCO3 in 80 ml MilliQ water
SECTION: Stock solution 4 preparation
6.2. Fill to 100 ml using MilliQ water
SECTION: Stock solution 4 preparation
6.3. Filter sterilize the solution
SECTION: Micronutrient stock solution preparation
7.1. Add trace metal salts in the order they are listed to 100 ml of MilliQ water and wait for complete dissolution between each addition
SECTION: Micronutrient stock solution preparation
7.2. Filter sterilize the solution
SECTION: Rhamnose stock preparation
8.1. Dissolve 8.2 g of rhamnose in 40 ml MilliQ water
SECTION: Rhamnose stock preparation
8.2. Add MilliQ water to 50ml
SECTION: Rhamnose stock preparation
8.3. Filter sterilize the solution
SECTION: Copper sulfate stock preparation
9.1. Dissolve 25 mg CuS04*5H20 in 8 ml MiliQ water
SECTION: Copper sulfate stock preparation
9.2. Add to 10 ml using MilliQ water
SECTION: Copper sulfate stock preparation
9.3. Dilute 10 µl of this solution in 9.99 ml MilliQ water
SECTION: Copper sulfate stock preparation
9.4. Filter sterilize the solution
SECTION: BG11-PC overview
1.2. 
BG-11 is split into four stock solutions such that incompatible (i.e. because of low solubility) compounds at high concentration are avoided. Additionally, a minimal amount of HEPES buffer is added to ensure the same starting pH of the cultures.
The final medium is prepared by adding buffer and each stock solution to 500 ml autoclaved water measuring cylinder and filling it to a total of 1 liter.