Feb 09, 2023
Version 3
Terrific broth (TB) medium V.3
This protocol is a draft, published without a DOI.
- 1University of Oslo
Protocol Citation: Andreas Sagen 2023. Terrific broth (TB) medium. protocols.io https://protocols.io/view/terrific-broth-tb-medium-cn5yvg7wVersion created by Andreas Sagen
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: Working
We use this protocol and it's working
Created: February 08, 2023
Last Modified: February 09, 2023
Protocol Integer ID: 76696
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
Yeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
Step 2
TryptoneMerck Millipore (EMD Millipore)Catalog #T9410
Step 2
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Step 2
GlycerolMP BiomedicalsCatalog #194680
Step 9
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
Terrific broth base solution
Terrific broth base solution
20m
20m
Fill the bottle with 200 mL distilled water
Note
Consider making a larger batch of 5 bottles of base solution at once and use all Potassium phosphate solution and carbon solution at once to get 2.5 L of medium
Measure 11.8 g Yeast extract and 5.9 g Tryptone. Add 7.5 g for making agar plates
Materials:
Yeast ExtractEmd MilliporeCatalog #Y0875
TryptoneEmd MilliporeCatalog #T9410
AgarEmd MilliporeCatalog #A1296
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 00:05:00
5m
Add 100 mL Potassium phosphate buffer (1 M, 6.2 pH)
Protocol
NAME
Potassium phosphate buffer (1.0 M, pH 6.2)CREATED BY
Andreas Sagen
Adjust pH while mixing to 7.2 using concentrated sodium hydroxide
Add distilled water to a total of 400 mL
Autoclave liquid at 121 °C for 00:15:00
15m
Terrific broth carbon solution
Terrific broth carbon solution
Fill the bottle with 300 mL double-distilled water
Add 20 mL Glycerol and mix solution well
Materials:
GlycerolEmd MilliporeCatalog #194680
Adjust pH while mixing to 7.2 using concentrated sodium hydroxide
Add distilled water to a total of 500 mL
Autoclave liquid at 121 °C for 00:15:00 Autoclave liquid at 121 °C for 00:15:00
15m
500 mL Terrific broth
500 mL Terrific broth
Add 100 mL carbon solution to base solution
Aliquot medium in 100 mL units, and store refrigerated (4 °C )
Note
If there is no space to store refrigerated, keep sterile and store at room temperature