Jan 09, 2023

Public workspaceTerrific broth (TB) medium V.1

This protocol is a draft, published without a DOI.
  • 1University of Oslo
Andreas Sagen
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Protocol Citation: Andreas Sagen 2023. Terrific broth (TB) medium. protocols.io https://protocols.io/view/terrific-broth-tb-medium-cmdfu23n
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: January 06, 2023
Last Modified: January 14, 2023
Protocol Integer ID: 74887
Keywords: Terrific broth, TB, cloning, plasmid, recombinant, E. coli, Escherichia coli
Abstract
IBI’s Terrific Broth is used with Glycerol in cultivating recombinant strains of E. coli. Terrific broth is a highly enriched medium for improving yield in plasmid bearing E. coli. Recombinant strains have an extended growth phase in the medium. The addition of tryptone and yeast extract in the medium will allow higher plasmid yield per volume. Glycerol is used as a carbohydrate source in this formulation. Unlike glucose, glycerol is not fermented to acetic acid.
Guidelines
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.
Materials
Analytical scale, autoclave, bottle(s), weight vessel, LAF bench
Protocol materials
ReagentYeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
ReagentTryptoneMerck Millipore (EMD Millipore)Catalog #T9410
ReagentPotassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5379
ReagentPotassium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P8281
ReagentGlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7757
Safety warnings
You can mix Dextrose from the beginning with the other compounds, and autoclave together. While this is more time efficient and easier, it is important to take into account the possibility of toxic byproducts produced by the Millard reaction when autoclaving, producing Acrylamide, a probable human carcinogen (IARC Group 2A).
Furthermore, when removing autoclaved components, be sure to take care as these can be very hot. If using antibiotics, use sufficient PPE to protect yourself, as some can be toxic to humans.
Before start
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated
500 mL Terrific broth
500 mL Terrific broth
20m
20m
Create a base solution consistent of Tryptone, Yeast extract, Potassium phosphate monobasic and Potassium phosphate dibasic in a Amount500 mL Reagent Bottle
Fill the bottle with Amount300 mL double-distilled water
1m
Measure Amount11800 mg Yeast extract, Amount5900 mg Tryptone, Amount1100 mg Potassium phosphate monobasic and Amount4700 mg Potassium phosphate dibasic Powdered compounds:
ReagentYeast ExtractEmd MilliporeCatalog #Y0875
ReagentTryptoneEmd MilliporeCatalog #T9410
ReagentPotassium phosphate monobasicEmd MilliporeCatalog #P5379
ReagentPotassium phosphate dibasicEmd MilliporeCatalog #P8281
5m
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for Duration00:05:00
10m
Adjust pH while mixing to Ph7.2 using concentrated sodium hydroxide
2m
Autoclave liquid at Temperature121 °C for Duration00:15:00
30m
Create a carbon solution consistent of glycerol in a Amount100 mL Reagent Bottle
Fill the bottle with Amount60 mL double-distilled water
1m
Add Amount4 mL Glycerol and mix solution well

Liquid compounds:
ReagentGlycerolEmd MilliporeCatalog #G7757
1m
Adjust pH while mixing to Ph7.2 using concentrated sodium hydroxide
2m
Add double-distilled water to a total of Amount100 mL
1m
Autoclave liquid at Temperature121 °C for Duration00:15:00
15m
After having prepared a base solution and carbon solution, combine the sterile solutions. All steps from this point take place in a LAF bench, to prevent contamination
Note
Remember allow flow to stabilize for minimum 15 minutes before use and clean LAF bench before use to prevent contamination of medium stock

Add Amount100 mL carbon solution to base solution, and mix well
1m
Add sterile water to a total of Amount500 mL
Note
Cool to 50°C and supplement with antibiotics as appropriate

1m