Aug 22, 2025

Public workspacePreparation of Auto-induction Terrific Broth for Protein Expression

DOI
https://dx.doi.org/10.17504/protocols.io.261gek5pjg47/v1
  • Yoon YL Lee1,
  • Amro Hamdoun1
  • 1Scripps Institution of Oceanography at UC San Diego
Yoon Lee
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DOI: https://dx.doi.org/10.17504/protocols.io.261gek5pjg47/v1
Protocol Citation: Yoon YL Lee, Amro Hamdoun 2025. Preparation of Auto-induction Terrific Broth for Protein Expression . protocols.io https://dx.doi.org/10.17504/protocols.io.261gek5pjg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 21, 2025
Last Modified: August 22, 2025
Protocol Integer ID: 225230
Keywords: induction terrific broth for protein expression, 10x terrific broth without phosphate, induction terrific broth, 10x terrific broth, 100x ammonium sulfate, 100x magnesium sulfate, 10x sodium phosphate, potassium phosphate, preparation of auto, protein expression, reagent, mgso4, phosphate, glucose, default scale for this protocol
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Abstract
The following is the default scale for this protocol for each reagent:

10X Terrific Broth without phosphates: 3 L
100X Magnesium sulfate (MgSO4, 125 mM): 1 L
100X Ammonium sulfate ((NH4)2SO4, 2.5 M): 1 L
20X Potassium phosphate (KH2PO4, 960 mM): 1 L
10X Sodium phosphate (Na2HPO4, 500 mM): 1 L
100X D-Glucose (C6H12O6, 1.60 M): 1 L
10X α-Lactose (C12H24O12, 222 mM): 1 L
50% Glycerol (C3H8O3, 6.84 M, 630 g/L): 1 L
1X Auto-induction Terrific Broth (made fresh from stocks per production run): 1 L

If you change the scale of the protocol through protocol.io's scaling feature when running the protocol, the scaled values are as follows:

10X Terrific Broth without phosphates: Amount3 L
100X Magnesium sulfate (MgSO4, 125 mM): Amount1 L
100X Ammonium sulfate ((NH4)2SO4, 2.5 M): Amount1 L
20X Potassium phosphate (KH2PO4, 960 mM): Amount1 L
10X Sodium phosphate (Na2HPO4, 500 mM): Amount1 L
100X D-Glucose (C6H12O6, 1.60 M): Amount1 L
10X α-Lactose (C12H24O12, 222 mM): Amount1 L
50% Glycerol (C3H8O3, 6.84 M, 630 g/L): Amount1 L
1X Auto-induction Terrific Broth (made fresh from stocks per production run):Amount1 L

The values for all components throughout the protocol should scale accordingly as well. The changes to the suggested container sizes may not make sense, so choose your container appropriately when changing the scale of the protocol. You can see this part of the abstract again by switching to the "Abstract" tab when running the protocol.

Protocol materials
ReagentMagnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1880-500G
ReagentAmmonium sulfateSanta Cruz BiotechnologyCatalog #sc-29085
ReagentPotassium Phosphate, MonobasicSanta Cruz BiotechnologyCatalog #sc-203211A
Reagentα-Lactose monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2643-500G
ReagentD-glucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021-1KG
ReagentSodium phosphate dibasic heptahydrateSanta Cruz BiotechnologyCatalog #sc-203402A
Reagent1000x Trace Metals Mixture. 1000mL, SterileTeknovaCatalog #T1001
ReagentYeast ExtractIBI ScientificCatalog #IB49162
ReagentTryptoneApexBio TechnologyCatalog #20-251
Troubleshooting
10X Terrific Broth without phosphates and glycerol
2h 55m
Prepare a Amount4 L beaker to mix broth components. Use a bottle for which you have verified the graduations are reliable.
5m
Using a Amount2 L graduated cylinder, measure Amount2 L of Milli-Q/deionized water, and pour the water into the beaker.
Place the beaker on a hot plate that is set to Temperature80 °C .
Let the water reach at least Temperature70 °C .
10m
Temperature
Measure and mix dry components with water to obtain a final volume of Amount3 L .
45m
Mix
Temperature
On a balance, measure Amount720 g of ReagentYeast ExtractIBI ScientificCatalog #IB49162 in a Amount1 L beaker.

Slowly add the yeast extract powder to the Amount4 L beaker with water until everything has dissolved. Stir using a reagent spatula as you add the powder to prevent clumping.
On a balance, measure Amount360 g of ReagentTryptoneApexBio TechnologyCatalog #20-251 in a Amount1 L beaker.

Slowly add the tryptone powder to the Amount4 L beaker with water until everything has dissolved. Stir using a reagent spatula as you add the powder to prevent clumping.
Top-up the beaker to Amount3000 mL with Milli-Q/deionized water.
Transfer the media to a Amount5 L glass media bottle using an appropriately sized funnel.
Close the bottle, leaving the cap slightly ajar, and affix autoclave tape to the cap and bottle.
Place the bottle of media in an autoclave pan filled with a 4 cm layer of water, and autoclave the media at Temperature121 °C liquid cycle for Duration00:55:00 .
55m
After autoclaving is complete, let the bottle cool to TemperatureRoom temperature toTemperature40 °C before handling.
Note
Do not tighten the bottle cap while cooling.

40m
Under an open flame, make single use aliquots of Amount100 mL in sterile Amount125 mL PET bottles.
Note
Keeping the base 10X Terrific Broth separately aliquoted out in single-use bottles allows for flexibility on how it might be used by different people across the lab (agar plates, smaller plasmid production runs, etc.). It also helps prevent potential contamination. Adjust aliquoting as needed for your lab.



10m
Optional
On 1.375" x 1.375" thermal labels, label each bottle with the following information:

Base 10X Terrific Broth- No phosphates, no glycerol
120g/L Tryptone
240g/L Yeast extract
Store at room temperature.
Sterile, autoclaved.
Date of manufacture: YYYYMMDD


100X Magnesium sulfate (MgSO4, 125mM)
15m
Prepare a Amount2 L beaker to mix components.

Using a Amount1 L graduated cylinder, measure Amount700 mL of Milli-Q/deionized water, and pour the water into the beaker.

Add a magnetic stir bar to the beaker and turn on the magnetic stir plate.
On a balance, measure Amount30.8 g ReagentMagnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1880-500G .

Slowly add the powder to the Milli-Q/deionized water until everything has dissolved.
Pour the solution back into a Amount1 L graduated cylinder.
Top up the volume to Amount1 L with Milli-Q/deionized water.
Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

100X Magnesium sulfate (MgSO4, 125mM)
30.8g/L Magnesium sulfate heptahydrate
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
100X Ammonium sulfate ((NH4)2SO4, 2.5M)
15m
Prepare a Amount2 L beaker to mix components.

Using a Amount1 L graduated cylinder, measure Amount700 mL of Milli-Q/deionized water, and pour the water into the beaker.

Add a magnetic stir bar to the beaker and turn on the magnetic stir plate.
On a balance, measure Amount330 g ReagentAmmonium sulfateSanta Cruz BiotechnologyCatalog #sc-29085 .

Slowly add the powder to the Milli-Q/deionized water until everything has dissolved.
Pour the solution back into a Amount1 L graduated cylinder.

Top up the volume to Amount1 L with Milli-Q/deionized water.

Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

100X Ammonium sulfate ((NH4)2SO4, 2.5M)
330g/L Ammonium sulfate
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
20X Potassium phosphate (KH2PO4, 960mM)
15m
Prepare a Amount2 L beaker to mix components.

Using a Amount1 L graduated cylinder, measure Amount700 mL of Milli-Q/deionized water, and pour the water into the beaker.

Add a magnetic stir bar to the beaker and turn on the magnetic stir plate.
On a balance, measure Amount130 g ReagentPotassium Phosphate, MonobasicSanta Cruz BiotechnologyCatalog #sc-203211A .

Slowly add the powder to the Milli-Q/deionized water until everything has dissolved.
Pour the solution back into a Amount1 L graduated cylinder.

Top up the volume to Amount1 L with Milli-Q/deionized water.

Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

20X Potassium phosphate (KH2PO4, 960mM)
130g/L Potassium phosphate, monobasic
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
10X Sodium phosphate (Na2HPO4, 500mM)
15m
Prepare a Amount2 L beaker to mix components.

Using a Amount1 L graduated cylinder, measure Amount900 mL of Milli-Q/deionized water, and pour the water into the beaker.

Add a magnetic stir bar to the beaker and turn on the magnetic stir plate.
On a balance, measure Amount134 g ReagentSodium phosphate dibasic heptahydrateSanta Cruz BiotechnologyCatalog #sc-203402A .

Slowly add the powder to the Milli-Q/deionized water until everything has dissolved.
Pour the solution back into a Amount1 L graduated cylinder.

Top up the volume to Amount1 L with Milli-Q/deionized water.

Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

10X Sodium phosphate (Na2HPO4, 440mM)
134g/L Sodium phosphate, dibasic
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
100X D-Glucose (C6H12O6, 1.60M)
15m
Prepare a Amount2 L beaker to mix components.

Using a Amount1 L graduated cylinder, measure Amount700 mL of Milli-Q/deionized water, and pour the water into the beaker.

Add a magnetic stir bar to the beaker and turn on the magnetic stir plate.
On a balance, measure Amount290 g ReagentD-glucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021-1KG .

Slowly add the powder to the Milli-Q/deionized water until everything has dissolved.
Pour the solution back into a Amount1 L graduated cylinder.

Top up the volume to Amount1 L with Milli-Q/deionized water.

Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

100X D-Glucose (C6H12O6, 1.60M)
290g/L D-glucose
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
10X α-Lactose (C12H24O12, 222mM)
15m
Prepare a Amount2 L beaker to mix components.

Using a Amount1 L graduated cylinder, measure Amount800 mL of Milli-Q/deionized water, and pour the water into the beaker.

Add a magnetic stir bar to the beaker and turn on the magnetic stir plate.
On a balance, measure Amount80 g Reagentα-Lactose monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2643-500G .

Slowly add the powder to the Milli-Q/deionized water until everything has dissolved.
Pour the solution back into a Amount1 L graduated cylinder.

Top up the volume to Amount1 L with Milli-Q/deionized water.

Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

10X α-Lactose (C12H24O12, 222mM)
80g/L α-Lactose monohydrate
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
50% Glycerol (C3H8O3, 6.84M, 630 g/L)
15m
Using a Amount1 L graduated cylinder, measure Amount500 mL of Milli-Q/deionized water.
Pour the water into a Amount1 L bottle.

Place the Amount1 L bottle on a balance, and zero the balance with the bottle.

Pour 100% glycerol into the bottle until the balance reaches Amount630 g .

Close the bottle and mix by inverting until no glycerol schlieren is visible.
Filter sterilize using a 0.22μm bottle top filter.
On a 1.375" x 1.375" thermal label, label the bottle with the following information:

50% Glycerol (C3H8O3, 6.84M)
630 g/L Glycerol
Store at room temperature.
Sterile, 0.22μm filtered.
Date of manufacture: YYYYMMDD
1X Auto-induction Terrific Broth (made fresh from stocks per production run)
10m
Mix the following components in a sterile Amount1 L beaker. Open stock reagents under an open flame in a sterilized work area.

1. Amount100 mL 10X Terrific Broth without phosphates and glycerol (pour from 100 mL aliquot bottle)
2. Amount100 mL 10X α-Lactose
3. Amount100 mL 10X Sodium phosphate
4. Amount50 mL 20X Potassium phosphate
5. Amount10 mL 100X Magnesium sulfate
6. Amount10 mL 100X Ammonium sulfate
7. Amount10 mL 100X D-Glucose
8. Amount20 mL 50% Glycerol
9. Amount1 mL 1000X Antibiotic (100 mg/mL Carbenicillin, 100 mg/mL Ampicillin, 50 mg/mL Kanamycin)
10. Amount1 mL Reagent1000x Trace Metals Mixture. 1000mL, SterileTeknovaCatalog #T1001
Using a sterile Amount1 L graduated cylinder, measure Amount600 mL of Milli-Q/deionized water, and pour the water into the beaker to mix the components in the beaker.
Pour the contents of the beaker back into the Amount1 L graduated cylinder, and cover the cylinder until you are ready to use the media (within ~1 hour).


Expected result
The following describes the composition of the complete 1X Auto-induction Terrific Broth Amount1 L :

24g/L Yeast extract
12g/L Tryptone
684mM Glycerol (12.6mg/L, 1.0%)
1.25mM Magnesium sulfate (0.15g/L)
25mM Ammonium sulfate (3.3g/L)
48mM Potassium phosphate (6.5g/L)
50mM Sodium phosphate (7.1g/L)
16mM Glucose (2.9g/L)
22mM Lactose (7.6g/L)

Trace metals
83.35 µM FeCl3 (13.52mg/L)
15.73 µM MnCl2 (1.98mg/L)
3.67 µM CoCl2 (476µg/L)
17.84 µM ZnSO4 (2.88mg/L)
3.511 µM NiCl2 (455µg/L)
2.375 µM Na2MoO4 (489µg/L)
26.49 µM CaCl2 (2.94mg/L)
2.536 µM CuCl2 (341µg/L)
2.00 µM Na₂SeO₃ (346µg/L)
2.00 µM H3BO3 (124µg/L)