Preparation of SOB (Super Optimal Broth) Medium

Overview

SOB (Super Optimal Broth) medium is a nutrient-rich microbial growth medium, formulated by Douglas Hanahan in 1983 (Hanahan, 1983). SOB or its glucose-supplemented version SOC is often used during the transformation of E. coli competent cells with plasmids (added in transformed cells after heat shock/electroporation). It results in better survival and higher transformation efficiency as compared to regular LB medium. The original recipe, described by Hanahan (Hanahan, 1983) contains 2% Bacto-tryptone (w/v), 0.5% Bacto-yeast extract (w/v), 10 mM Sodium chloride (NaCl), 2.5 mM Potassium chloride (KCl), 10 mM Magnesium sulfate (MgSO4), and 10 mM Magnesium chloride (MgCl2).

SOB medium is prepared without Mg++ by dissolving 20 g bacto-tryptone, 5 g bacto-yeast extract, 2 ml of 5M sodium chloride, and 2.5 ml of 1M KCl in water to a final volume of ≈ 1000 ml (980 ml). Magnesium salts  (i.e., MgSO4 and MgCl2, 10 ml each of 1M MgSO4 and 1M MgCl2) are added to the medium after autoclaving to prevent any chemical reactions and precipitation of the medium components.

REQUIREMENTS

Reagents and solutions
Bacto-tryptone
Bacto-yeast extract
5M Sodium chloride (NaCl)
1M Potassium chloride (KCl)
1M Magnesium sulfate (MgSO4) (Sterile)
1M Magnesium chloride (MgCl2) (Sterile)
5N NaOH (for pH adjustment)
Deionized / Distilled water 

Equipment and disposable
Measuring cylinder
Erlenmeyer flask / Beaker
Hot plate / Magnetic stirrer with a heating device

Composition
2% (w/v) Bacto-tryptone
0.5% (w/v) Bacto-yeast extract
10 mM Sodium chloride
2.5 mM Potassium chloride (KCl)
10 mM Magnesium sulfate (MgSO4)
10 mM Magnesium chloride (MgCl2)

Objective
Preparation of 1000 ml SOB medium

PROCEDURE

Step 1: To prepare 1000 ml of SOB medium, weigh out 20 g bacto-tryptone, and 5 g bacto-yeast extract in a 2-litre Erlenmeyer flask/beaker. Add 800 ml deionized/Milli-Q water and mix it. The solution will appear translucent yellowish with undissolved media ingredients.

Tip:
◊ One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.

Precautions:
◊ Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute is dissolved in a solvent.
◊ Make sure no lumps are remaining after making the suspension of medium components.

Step 2: Dissolve all ingredients completely by heating to boiling while stirring.

Note:
◊ Medium with completely dissolved ingredients will appear as a transparent yellowish solution.

Precautions:
◊ Once all ingredients are dissolved and the medium appears transparent, stop the heating. Don’t unnecessarily heat the medium.
◊ Make sure to dissolve any residual powder sticking to the glass.

Step 3: Cool down the medium to room temperature. Add 2.5 ml of 1M KCl and mix. Adjust the pH 7.0 with 5N NaOH (~0.2 ml).

Note:
The SOB medium is not very highly buffered. The pH of the medium drops, as the growing culture reaches the near saturation phase.

Step 4: Adjust the volume to 980 ml with deionized / Milli-Q water. Mix it again.

Step 5: Transfer the medium to an autoclavable bottle or insert the cotton plug and cover the mouth Erlenmeyer flask with aluminum foil.

Step 6: Sterilize the solution by autoclaving for 20 minutes at 15 lb/in2 (1.05 kg/cm2) on liquid cycle.

Step 7: Let the solution cool down to room temperature. Add 10 ml of 1M MgSO4 and 10 ml of 1M MgCl2 aseptically and mix by shaking the bottle.

Tips:
◊ Antibiotics and nutritional supplements should be added only after the solution has cooled to 40°C or below. Many supplements, like antibiotics, are degraded at high temperatures.
◊ After autoclaving, all sterile solutions should be handled inside the laminar flow hood to maintain sterility.

Storage
The solution can be stored at room temperature for a few days. Keep the medium at 2 – 8 °C for longer storage.

Follow the table to prepare SOB medium of various volumes (100 ml, 500 ml, 1,000 ml and 10,000 ml).
Reagents / Volume100 ml500 ml1,000 ml10,000 ml
Bacto-tryptone2 g10 g20 g200 g
Bacto-yeast extract0.5 g2.5 g5 g50 g
Sodium chloride0.5 ml1 ml2 ml20 ml
1M KCl0.25 ml1.25 ml2.5 ml25 ml
1M MgSO41 ml5 ml10 ml100 ml
1M MgCl21 ml5 ml10 ml100 ml
WaterAdjust the final volume to 100 mlAdjust the final volume to 500 mlAdjust the final volume to 1000 mlAdjust the final volume to 10000 ml

REFERENCES:

Hanahan, 1983. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 166(4), 557-80. PMID-6345791; Full-Text Link: sciencedirect, citeseerx

Was this post helpful?

Author: admin

Leave a Reply

Your email address will not be published. Required fields are marked *