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 H₂O 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

LB: Luria-Bertani

Rich media used for routine culture of E. coli and other bacteria at high cell densities.

1L	5L	Component
10 g	50 g	Tryptone
5 g	25 g	Yeast Extract
10 g	50 g	NaCl

Add dH₂O to final volume. Autoclave. If mixing up large batches and aliquoting, be sure to add exact volumes to final media bottles so that they will be ready for addition of antibiotic to known concentrations.

Note: This recipe is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

Expected results: E. coli strains grow to 5×10⁹ cells/ml final density in LB.

Variant: 0.1×LB is made with 1 g Tryptone, 0.5 g Yeast Extract, and 10 g NaCl per liter. (It is 0.1× in the nutrients, but not the salt!)

Included topic: ProtocolsRecipesLuriaBertani

DM: Davis Minimal

1L	5L	Component	MW
7 g	35 g	Potassium Phosphate (dibasic, trihydrate) HK2PO4 (H2O)3	MW 228.17
2 g	10 g	Potassium Phosphate (monobasic) H2KPO4	MW 136.07
1 g	5 g	Ammonium Sulfate (NH4)2 SO4	MW 132.08
0.5 g	2.5 g	Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2	MW 294.10

Add dH₂O to final volume and autoclave.

After autoclaving add the following stock solutions:

1L	5L	Component
1.0 ml	5 ml	10% Magnesium Sulfate MgSO4
1.0 ml	5 ml	0.2% Thiamine (vitamin B1) (filter sterilized)

Final nutrient concentrations:

67.4 mM	Phosphate (PO43-)
8.38 mM	Sulfate (SO42-)
15.1 mM	Ammonium (NH4)
5.1 mM	Sodium (Na+)
98.1 mM	Potassium (K+)
0.83 mM	Magnesium (MG2+)
1.7 mM	Citrate
139 µM	Glucose (in DM25)

Included topic: ProtocolsRecipesDavisMinimal

NZY+ Broth

1L	Component
10 g	NZ Amine (casein hydrolysate)
5 g	Yeast Extract
5 g	NaCl

Add dH₂O to final volume. Adjust pH to 7.5 using NaOH (Requires ~350 µl of 10 N). Autoclave. Then add.

1L	Component
12.5 ml	1 M MgCl2
12.5 ml / 15.0 ml	1 M / 10% (w/v) MgSO4 FW = 120.3 g/mol
40 ml	10% (w/v) glucose

Included topic: ProtocolsRecipesNZY+Broth

M9 Minimal

Make 5× M9 salts:

1L	Component
64 g	Sodium phosphate (dibasic, 7 hydrate) Na2HPO4•H2O
15 g	Potassium phosphate (monobasic) KH2PO4
2.5 g	Sodium chloride, NaCl
5 g	Ammonium chloride NH4Cl

Add dH₂O to 1 liter. Autoclave.

Combine the following ingredients (all pre-sterilized):

1L	Component
200 ml	5× M9 salts
2 ml	1M Magnesium sulfate MgSO4
0.1 ml	1 M Calcium chloride CaCl2
40 ml	10% (w/v) glucose (or other carbon source)

Add sterile dH₂O to 1 liter. Autoclave.

To make M9 plates add the items above to 16g of sterilized agar.
If making less than 1 liter use 8g of agar per 500ml of solution.

Included topic: ProtocolsRecipesM9Minimal

R2A

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: Super Optimal Broth

1L	Final [ ]	Component
5 g	0.5%	yeast extract
20 g	2.0%	tryptone
0.5 g	10 mM	NaCl
0.186 g	2 mM	KCl
2.4 g	20 mM	MgSO4

Add 800 ml of water and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add H₂O to a final volume of 960 ml. Autoclave.

Included topic: ProtocolsRecipesSOB

SOC: Super Optimal Broth with Glucose

Used for recovery of cells after electroporation or heat shock transformation.

1L	Final [ ]	Component
5 g	0.5%	yeast extract
20 g	2.0%	tryptone
0.5 g	10 mM	NaCl
0.186 g	2 mM	KCl
2.4 g	20 mM	MgSO4

Add 800 ml of water and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add H₂O to a final volume of 960 ml. Autoclave. When solution has cooled to 50°C (cool enough to touch with bare hands for a few seconds), add 40 ml of autoclaved 10% glucose.

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

LB: Luria-Broth

Combine in a 2 L flask the following:

1.5L	Component
15 g	Tryptone
7.5 g	Yeast Extract
15 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 1.5 L. Autoclave.

Note: This is different from "LB" = "Luria Broth" media, which has only 5 g NaCl per liter!

To make "soft" agar for phage studies, use only 7 g agar per liter.

Included topic: ProtocolsRecipesLuria-Broth

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

Combine in a 2 L flask:

1.5L	Component
15 g	Tryptone
1.5 g	Yeast Extract
7.5 g	NaCl
24 g	Agar
1.5 ml	5% Antifoam

Add dH₂O to 1.3 liter.

Combine in a 500 ml flask:

1.5L	Component
6 g	Sugar (arabinose = TA, maltose = TM, lactose = TL)

Add dH₂O to 0.2 liter.

Autoclave sugar and media separately. Total combined volume is 1.5 liters.

Combine and add:

1.5L	Component
1.5 ml	TTC (5%)

Source: Lenski Lab Protocol

Included topic: ProtocolsRecipesTetrazoliumSugar

MG: Minimal Glucose, aka DM: Davis Minimal

Note: When making these plates it is necessary to prepare and autoclave the 3 main parts (salts, agar, and sugar) separately.

1L	1.5L	Component	MW
7 g	10.5 g	Potassium Phosphate (dibasic, trihydrate) HK2PO4 (H2O)3	MW 228.17
2 g	3 g	Potassium Phosphate (monobasic) H2KPO4	MW 136.07
1 g	1.5 g	Ammonium Sulfate (NH4)2 SO4	MW 132.08
0.5 g	0.75 g	Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2	MW 294.10
333 ml	500 ml	dH2O

Autoclave.

Next, prepare the agar:

1L	1.5L	Component
16 g	24 g	Agar
1 ml	1.5 ml	Antifoam (5%)
333 ml	500 ml	dH2O

Autoclave.

Next, prepare the sugar (0.2%):

1L	1.5L	Component
4 g	6 g	Glucose (or other sugar)
333 ml	500 ml	dH2O

Autoclave.

After the 3 parts have been autoclaved, combine the contents of the three flasks together and add the following stock solutions:

1L	1.5L	Component
1.0 ml	1.5 ml	10% Magnesium Sulfate MgSO4 (autoclaved)
1.0 ml	1.5 ml	0.2% Thiamin (vitamin B1) (filter sterilized)

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

SOB: Super Optimal Broth Plates

1L	Final [ ]	Component
5 g	0.5%	yeast extract
20 g	2.0%	tryptone
0.5 g	10 mM	NaCl
0.186 g	2 mM	KCl
2.4 g	20 mM	MgSO4
16 g		Agar
1ml		Antifoam

Add 800 ml of water and dissolve components. Adjust to pH 7.5 prior to use with 1 M NaOH (approximately 25 ml). Add H₂O to a final volume of 960 ml. Autoclave.

Included topic: ProtocolsRecipesSOBplates

PA: Lac Papillation Agar

Make 10× Minimal A Salts.

10× Minimal A Salts
1L	Component	MW
105 g	Potassium Phosphate (dibasic, trihydrate) HK2PO4 (H2O)3	MW 228.17
45 g	Potassium Phosphate (monobasic) H2KPO4	MW 136.07
10 g	Ammonium Sulfate (NH4)2 SO4	MW 132.08
5 g	Sodium Citrate (trisodium, dihydrate) Na3C6H5O7 (H2O)2	MW 294.10
to 1L	dH2O

Autoclave. Salt solution can be stored at room temperature.

Agar Solution
1L	Component
15 g	Agar
to 590 ml	dH2O

Autoclave.

Sugar Solution
1L	Component
2 g	Glucose
to 400 ml	dH2O

Autoclave.

After autoclaving, combine agar and sugar and add:

1L	Component
100 ml	10× Minimal A Salts
6 ml	10% w/v (0.083M Mg2+) Magnesium Sulfate, anhydrous MgSO4 (autoclaved) MW 120.37
2.5 ml	0.2% w/v Thiamin (vitamin B1) (filter sterilized)
1 ml	500 mg/ml P-Gal (phenyl β-D-galactoside) (filter sterilized)
1 ml	40 mg/ml X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) (filter sterilized)

Note: The amounts added of some standard components (including agar) are slightly different for this recipe. Volumes have also been altered from the original Miller recipe to use the same stock solutions we have available for making DM plates. The final concentrations are 0.2% glucose, 0.05% P-Gal, and 0.004% X-Gal (w/v).

Sources:

1. Nghiem, Y., Cabrera, M., Cupples, C.G., Miller, J.H. (1988) The mutY gene : A mutator locus in Escherichia coli that generates G•C→T•A transversions. Proc. Natl. Acad. Sci. U.S.A. 85:2709-2713.
2. Miller J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Included topic: ProtocolsPapillationAgar