SOB Medium Preparation
Introduction
Materials
- Consumables
- tryptone
- yeast extract (yeastolate)
- NaCl
- bacto agar (optional)
- MgCl2 stock solution
- NaOH
- selectable component (e.g., antibiotics) (optional)
- Disposables
- weigh dishes/paper
- petri dishes (optional)
- Equipment
- media bottle
- pH meter
- autoclave
- balance
Procedure
Component Composition
A A | B B | |
1 1 | Final Volume (mL)* | 1000 |
A A | B B | |
1 1 | Component | Weight (g) |
2 2 | tryptone | 20 |
3 3 | yeast extract | 5 |
4 4 | NaCl | 0.5 |
5 5 | bacto agar (optional) | 15 |
A A | B B | C C | |
1 1 | Solution | Stock Concentration (M)* | Amount (mL) |
2 2 | MgCl2 | 2 | 5 |
Preparation
- Add ~80% of the final volume of dH2O to a sterile media bottle.
Generally, the capacity of the bottle should be greater than (not equal to) the final volume of media to be prepared in order to prevent the media from boiling over during the autoclave step.
Ensure you have a volumetric flask measuring your desired final volume.
- Adjust the final volume in the table above and add the calculated amounts of tryptone, yeast extract, and NaCl to the dH2O.
CRITICALDo not add agar at this point if you are preparing solid medium.
- Seal the cap of the media bottle and shake until components are completely dissolved.
- Neutralize (pH = 7.0) the solution using NaOH.
Approximately 1-2mL of 1N NaOH should be expected.
- Transfer the solution to a sterile volumetric flask and adjust the final volume using dH2O, then return it to the media bottle.
Ensure the solution is well mixed before returning it to the media bottle.
- SOLID MEDIUM ONLY Add the appropriate amount of agar to the solution.
CRITICALBacto agar does not need to dissolve at this point.
Note: The density of agar is ~1g/mL, it should only increase the final volume by 1.5%, which is negligible.
- Apply autoclave tape to the bottle and autoclave on liquids cycle. Read step 8.
CRITICALLiquids cycle.
- If adding selectable components or preparing solid medium, begin warming a water bath to 55°C.
SOLID MEDIUM ONLY Begin labeling approximately 1 plate per15mL of medium.
- LIQUID MEDIUM ONLY If you are not adding selectable components, go to step 14.
- Allow solution to cool to 55°C in water bath.
- If adding selectable components, add them at this point.
CRITICAL Ensure solution is well mixed.
- SOLID MEDIIUM ONLY Add the proper amounts of sterile magnesium chloride (10mM final).
- SOLID MEDIUM ONLY Remove from waterbath and pour approximately 15mL into each labeled petri dish and allow to cool to room temperature.
CRITICAL Ensure entire bottom of dish is covered.
- SOLID MEDIUM ONLY Once media has gelled, replace the dishes inverted into their respective sleeve for storage.
CRITICALSolid media should always be stored/incubated in the inverted position.
- Store media at 4°C.
- CRITICALLIQUIDS ONLY Before use, be sure to add the proper amounts of sterile magnesium chloride (10mM final).
Values can be re-calculated on a per-use basis by adjusting the final volume above.
This protocol is adapted from Sambrook & Russell for the preparation of SOB medium. It describes preparation of both liquid and 1.5% solid agar media.
It is important to note that liquid SOB is stored without the required magnesium chloride and the magnesium chloride should be added to a final concentration of 10mM immediately before use.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3 edn. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. pp. A2.3