Media & Recipes

Dietary requirements for your little ones and other needed recipes.

The recipes on this page have been developed for or adapted to suit the needs of projects in our lab, primarily with a focus on bacteria, such as Escherichia coli. Each will make a defined volume, but they can be scaled up or down as needed.

Use the Table of Contents on the right to quickly navigate to the desired recipe.

ddH2O refers to ultra-pure water. ddH2O should be your go-to for all media and enzymatic reactions. Never use DI or standard tap water. Ultra-pure water can be found by the ice machine in the shared instrumentation space (Lab 241).

LB (Lysogeny Broth)

Also known as Luria-Bertani (LB) broth, one of the most widely used medium for the growth of bacteria (Bertani, et al., 1951). Formulation per one liter (add the following components to 1 L of ddH2O):

1. 10 g tryptone

2. 10 g NaCl

3. 5 g yeast extract

We order a prepared LB mix. Add 25 g per liter ddH2O of Difco LB Broth, Miller (Luria-Bertani).

SOC (Super Optimal Broth)

Super Optimal Broth (SOB medium) is a nutrient-rich bacterial growth medium used for microbiological culture, generally of Escherichia coli. It was developed by Douglas Hanahan in 1983 and is an adjusted version of the commonly used LB media (lysogeny broth). Super Optimal Broth with Catabolite Repression (SOC) is SOB with added glucose. SOC Medium is used in the final step of bacterial cell transformation to obtain maximal transformation efficiency of E. coli.

First prepare 20% (20 g / 100 mL) glucose solution by adding 100 g glucose to 500 mL ddH2O, and filter sterilize using a 500 mL Nalgene® Rapid-Flow™ Filter Unit. This solution can be stored at your bench. Make sure to use aseptic technique when using it.

Next, add 28.08 g SOC Broth Base to 980 mL ddH2O. Autoclave. Allow to cool to room temperature, and aseptically add 20 mL filter sterilized 20% glucose solution.

Since SOC medium is super rich, it's extremely prone to contamination. You can aliquot out 15 or 50 mL volumes of SOC in VWR tubes so that if one tube gets contaminated, you can discard it, and use another, and you won't contaminate the entire SOC stock that you prepared!

LB Agar (Plates)

The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within. The addition of an antibiotic to this gel allows for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene.

1. Add 15 g Bacto Agar to your LB recipe before autoclaving.

2. Add a medium sized magnetic stir bar. Add foil and autoclave tape per usual. Autoclave.

3. Transfer LB agar bottle to a hot plate and turn the magnetic stirring on to homogenize the liquid media. Allow the LB agar to cool to roughly 60°C (or until it's just cool enough to touch for a few seconds, but still warm).

4. Add your antibiotic(s) if you are preparing selective media. Allow one minute for mixing.

5. Using aseptic technique, pour the molten LB agar into Petri dishes at your bench (keep the sleeve).

6. Let the plates cool for at least 6 hours, or until the LB agar is solid.

7. Store the sleeved plates (label date and antibiotics used with VWR tape) in the 4°C fridge.

Addgene has a very detailed tutorial with YouTube video here.

Recommendations

1. Pour just enough molten LB agar into each Petri dish to cover the entire area. In some cases you may want to pour thicker plates; for example, you have long incubation times (i.e. greater than 24 hours).

2. You can use a Pipet Aid (auto-pipette) with serological pipette to collect and dispense LB agar.

3. Array the poured plates in a grid rather than stacking them to increase cooling speed.

4. You can individually label your plates with the antibiotic used, date, and your initials. For example, I might write "Amp50 ACR". This helps when you are using multiple antibiotics.

Selective plates vary in mileage. LB agar plates with ampicillin should be used within two weeks. Plates with other antibiotics can typically be used within one month without reduced effectiveness.

Antibiotics

Stock Concentrations

• Chloramphenicol (Cm): 50 mg/mL in ethanol

• Kanamycin (Kan): 50 mg/mL in water

• Streptomycin (Strep): 50 mg/mL in water

• Ampicillin (Amp): 50 mg/mL in water

Filter sterilize and aliquot ~800 uL into autoclaved Eppendorf tubes. All stocks should be stored at -20C.

Working Concentrations

Chloramphenicol: 5-50 μg/mL in ethanol 50 μg/mL for strong selection in rich media (important when cloning) 5 μg/mL for use in M9 minimal media

Kanamycin: 50 μg/mL in water 5-10 μg/mL for use in M9 media or when transforming two plasmids

Streptomycin: 50 μg/mL in water

Ampicillin:50 μg/mL in water

Tips & Tricks:

• Ampicillin is very light and heat sensitive. To be safe, always use Amp plates within 24 hours of making them.

• Best-use shelf life of Cm, Kan, and Strep plates: 1 week max when stored at 4 deg C

Inducers

Anhydrotetracycline (aTc)

Prepare a 1,000 times stock solution: 100 ug/mL solution in 50% ethanol. Store in a foil-covered tube at -20 °C. Alex: Inducing with 0.625 ng/mL, I prepped a 600 u/mL stock and did 2 x1000 serial dilutions.

TAE buffer

Instructions for prep are written on top of TAE carboy. Empty carboy. To 300 mL 50x TAE add ddH2O to make volume up to 15 L.

Ladder (for gel electrophoresis)

* 20 uL ladder stock
* 813 uL ddH2O
* 167 uL loading dye

M9 Minimal Media (MM) with 0.4% w/v Glucose

Reagent	Stock Concentration	Add (1 L)	Add (500 mL)
ddH2O	-	700	350
M9 salts solution	5x autoclaved	200	100
Glucose	20% sterilized	20	10
Leucine (1)	1 g/100 mL autoclaved	5	2.5
Thiamine (2)	1 g/100 mL (1% w/v)	34	17
MgSO4	1 M autoclaved	2	1
CaCl2	1 M autoclaved	0.1	0.05
Biotin (3)	0.25 mg/mL	1	0.5
NaOH	1 M	12-15 (4)	~5 (4)
HCl	1 M	(5)	(5)
ddH2O	-	to 1 L	to 500 mL

Filter sterilize. pH should be at 7.4 after titrating with NaOH and HCl.

1. DH10B is auxotrophic for Leucine

2. E. coli D5-alpha is auxotrophic for Thiamine

3. Only add Biotin if you’re working with EcNR2!

4. Titrate room temperature NaOH 1 mL at a time until you hit target pH of 7.4.

5. If you go over 7.4, use HCl to bring pH back down.

Tet/SacB counter-selection medium

Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli Xin-tian Li, Lynn C. Thomason, James A. Sawitzke, Nina Costantino and Donald L. Court

"The Tet/SacB counter-selection agar that was developed here contains, per liter, 15 g of Difco agar, 4 g of tryptone, 4 g of yeast extract, 8 g of NaCl, 8 g of NaH2PO4·H2O, 0.11 g ZnCl2, 24 mg fusaric acid and 60 g sucrose. The agar, tryptone and yeast extract were autoclaved in a 400-ml volume with water. The NaCl and NaH2PO4·H2O were mixed and autoclaved in a 400-ml volume with water. Sucrose at 60 g in 100 ml was autoclaved. The molten agar mix, salt mix and sucrose were combined together after autoclaving. The fusaric acid (Sigma) was stored at 48 mg/ml in ethanol at -20C in a lightproof container. The ZnCl2 was stored in water at 25 mM after filter sterilizing; note that higher concentrations precipitate. Fusaric acid (0.5 ml) and ZnCl2 (32 ml) were added to the molten agar mixture after cooling to 55C. The final volume was brought to 1 liter with sterile H2O.

Petri plates (100 mm) were poured so that each contained a 40-ml volume of the final molten agar mixture. The large volume ensures that the plates will remain hydrated overseveral days of incubation at 42C. After hardening, the plates were placed in their original plastic bags, wrapped in aluminum foil and stored at 4C. We emphasize the importance of precisely following these procedures as variations can dramatically affect the selection."

Preparation steps 1. 15 g Difco agar, 4 g tryptone, 4 g yeast extract add to 400 mL volume with ddH2O (autoclave) 2. 8 g NaCl, 8 g NaH2PO4·H2O add to 400 mL volume with ddH2O (autoclave) 3. 60 g sucrose add to 100 mL ddH2O (filter sterilize) 4. Fusaric acid prep aliquots at 48 mg/mL in EtOH (store in -20C) 5. Prepare 25 mM ZnCl2 in ddH2O (filter sterilize) 6. Combine molten (1-3) after autoclaving 7. Add 0.5 mL fusaric acid and 32 mL ZnCl2 to the molten mixture after cooling to 55C 8. Bring final volume to 1 liter with ddH2O