Media Recipes

Liquid Media

Sterile Saline

Purpose: Used make dilutions of viable cells for plating or transfer to new media.

This saline concentration of 0.85% w/v (145 mM) is suitable for diluting E. coli before transferring to new media or plating on agar.

1L	5L	Component
8.5 g	42.5 g	NaCl

Add dH₂O to final volume. Autoclave before use. Prepare large batches in 6 L flask and then aliquot out into bottles. It is not important to measure out exact volumes when dividing a large batch among bottles.

Included topic: ProtocolsRecipesSaline

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Included topic: ProtocolsRecipesLuriaBertani

Warning: Can't find topic Lab.ProtocolsRecipesDavisMinimal

Included topic: ProtocolsRecipesDavisMinimal

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Included topic: ProtocolsRecipesNZY+Broth

M9 Minimal Medium

1L	Component
6 g	Sodium phosphate dibasic (anhydrous), Na2HPO4
3 g	Potassium phosphate monobasic, KH2PO4
0.5 g	Sodium chloride, NaCl
1 g	Ammonium chloride, NH4Cl

Add dH₂O to 1 liter. Autoclave.

After media is autoclaved add the following sterile ingredients:

1L	Component
1 ml	1M Magnesium sulfate, MgSO4
1 ml	0.1M Calcium chloride CaCl2
10 ml	10% (w/v) glucose (or other carbon source)

Final composition
93.0 mM	Sodium (Na+)
22.1 mM	Potassium (K+)
18.7 mM	Ammonium (NH4)
1.0 mM	Calcium (Ca2+)
0.1 mM	Magnesium (Mg2+)
29.2 mM	Chloride (Cl-
0.1 mM	Sulfate (SO42-)
42.2 mM	Phosphate (PO43-)

Included topic: ProtocolsRecipesM9Minimal

R2A

R2A is a medium that can be used to grow a wide variety of soil microbes.

1L	5L	Component
0.5 g	2.5 g	Yeast Extract
0.5 g	2.5 g	Proteose Peptone No. 3
0.5 g	2.5 g	Casamino Acids
0.5 g	2.5 g	Dextrose
0.5 g	2.5 g	Soluble Starch
0.3 g	1.5 g	Sodium Pyruvate
0.3 g	1.5 g	Dipotassium Phosphate
0.05 g	0.25 g	Magnesium Sulfate

Add dH₂O to final volume and autoclave.

If making R2A solid media also add 15 g of agar and 1 ml of 5% antifoam per liter.

Included topic: ProtocolsRecipesR2A

SOB/SOC: Super Optimal Broth

For SOB:

200mL	250mL	1L	Final [ ]	Component
1 g	1.25 g	5 g	0.5%	yeast extract
4 g	5 g	20 g	2.0%	tryptone
0.12 g	0.125 g	0.5 g	10 mM	NaCl
0.037 g*	0.042 g*	0.186 g	2 mM	KCl
0.48 g	0.6 g	2.4 g	20 mM	MgSO4 (anhydrous)

*Don't measure; it's just a pinch.

Adjust to pH 7.5 with 1M NaOH.

Note: 200 ml of SOC makes 25 vials of 8 ml each.

For 1 L: Add 800 ml of dH₂O and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add dH₂O to a final volume 1 L.

Autoclave.

For SOC (=SOB+glucose)

Bulk scale: Make SOB, except add dH₂O to a final volume of 960 ml instead of 1 L. Autoclave. When solution has cooled to 50°C (cool enough to touch with bare hands for a few seconds), add 40 ml of sterile 10% (w/v) glucose. (That's a final concentration of 0.4% glucose or about 20 mM.)

Smaller scale: To achieve a 20 mM concentration of glucose for a known final volume n of SOC, you can calculate the amount of 10% w/v glucose to be added as 0.036n. The amount of SOB to add is then 0.964n. Keep units constant, so if n is in µL, the final amounts will also be in µL and so on.

• Note: If you have a known volume of SOB x and want to know how much 10% w/v glucose to add to make SOC containing 20 mM glucose, simply divide x/0.964 to get your final volume n and subtract x from n to get the volume of 10% w/v glucose to add.

Generally when making stocks of SOC it is advisable to make smaller volume aliquots rather than larger ones. SOC media is extremely rich and can contaminate easily particularly after glucose is added. Additionally, smaller volumes tend to be pulled from it repeatedly with pipettes, which increases the chances of contamination over time.

Note: Some recipes for SOB use 10 mM MgSO₄ and 10 mM MgCl.

Included topic: ProtocolsRecipesSOB

Warning: Can't find topic Lab.ProtocolsRecipesSOC

Included topic: ProtocolsRecipesSOC

TGY Medium

1L	5L	Component
5 g	25 g	Pancreatic digest of casein
5 g	25 g	Yeast Extract
1g	5 g	Glucose
1 g	5 g	K2HPO4

Add dH₂O to final volume and autoclave.

Included topic:ProtocolsRecipesTGY

DR: Defined minimal media for D. radiodurans

250 ml	500 ml	Component
50 ml	100 ml	5x M9 salts
250 ul	500 ul	5mM MnCl2
250 ul	500 ul	0.8M MgCl2
250 ul	500 ul	0.18 M CaCl2
2.5 ml	5 ml	Syringe filtered Vitamins Mix
500 ul	1 ml	Syringe filtered Amino Acid mix (see below)

Add dH₂O to final volume.
Note: All components are either pre-sterilized or syringe filtered so the only things that need to be autoclaved are the receiving bottle and the added water.

To prepare the amino acids:

Amount	Amino Acid
1 g	L-Cys
0.5 g	L-His
0.5 g	L-Met

Dissolve these amino acids in 40 ml of dH₂O

Included topic:ProtocolsRecipesDR

Solid Media

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Included topic: ProtocolsRecipesLuria-Broth

TA: Tetrazolium Sugar (TA, TM, TL, ...)

Combine in a 1 L flask:

500 mL	Component
5 g	Tryptone
500 mg	Yeast Extract
2.5 g	NaCl
8 g	Agar
500 µL	5% Antifoam

Add dH₂O to 433.33 mL.

Combine in a 125 ml flask:

500 mL	Component
5 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 66.67 mL.

Cover each flask with foil, and autoclave sugar and media separately. Total combined volume will be 500 mL.

Combine the autoclaved sugar and media solutions and add:

500 mL	Component
500 µL	5% (w/v) Triphenyl Tetrazolium chloride (TTC), filter sterilized, stored at 4°C

Source: Lenski Lab Protocol

Included topic: ProtocolsRecipesTetrazoliumSugar

MG: Minimal Glucose agar, aka DM: Davis Mingioli agar

Note: When making these plates it is necessary to prepare and autoclave the 3 main parts (salts, agar, and sugar) separately. Compounds that inhibit growth are produced when agar and phosphate or phosphate and glucose are autoclaved together.

500 mL	1.0L	Component
2.65 g	5.3 g	Potassium Phosphate (dibasic) K2HPO4
1 g	2 g	Potassium Phosphate (monobasic) KH2PO4
0.5 g	1 g	Ammonium Sulfate (NH4)2 SO4
0.25 g	0.5 g	Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2
166.7 mL	333 mL	dH2O

Separately, prepare the agar:

500 mL	1.0 L	Component
8 g	16 g	Agar
500 µL	1 mL	Antifoam (5%)
166.7 mL	333 mL	dH2O

Next, prepare the sugar solution. The final concentration is 0.4% w/v:

500 mL	1.0 L	Component
2 g	4 g	Glucose, arabinose, or other sugar
166.7 mL	333 mL	dH2O

Autoclave the 3 parts separately for a 40 minute sterilization time.

After the 3 parts have been autoclaved, combine the contents of the three flasks together while they are still warm add the following stock solutions:

500 mL	1.0 L	Component
500 µL	1.0 ml	10% MgSO4 (separately autoclaved stock)
500 µL	1.0 ml	0.2% Thiamine (vitamin B1) (filter sterilized stock refrigerated at 4ºC)

Source: Lenski Lab Protocol

Included topic: ProtocolsRecipesMinimalGlucose

MC: Minimal Citrate

Prepared the same as MG: Minimal Glucose with the following changes:

• No glucose
• 4.5 g/L Sodium Citrate (trisodium, dihydrate)

Included topic: ProtocolsRecipesMinimalCitrate

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Included topic: ProtocolsRecipesSOBplates