Jun 13, 2019

Public workspaceLB Media

DOI
https://dx.doi.org/10.17504/protocols.io.3idgka6
  • William Brydon1,
  • Sricharan Kadimi1
  • 1University of Connecticut
  • UConn iGEM
William Brydon
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DOI: https://dx.doi.org/10.17504/protocols.io.3idgka6
Protocol Citation: William Brydon, Sricharan Kadimi 2019. LB Media. protocols.io https://dx.doi.org/10.17504/protocols.io.3idgka6
Manuscript citation:
Taken from Benchling Protocol
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 in our group and it is working, though open to further improvement/modification.
Created: May 30, 2019
Last Modified: June 13, 2019
Protocol Integer ID: 23845
Keywords: bacterial culture, lb media lb, overnight culture, overnight cultures after transformation, media, luria, culture
Abstract
LB (Luria-Bertani) media is commonly used for bacterial culture. We use it for overnight cultures after transformations.
Materials
MATERIALS
ReagentYeast ExtractCatalog #Y1625
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentTryptoneFisher ScientificCatalog #BP1421-500
  • 1 Liter Glass Bottle
  • Autoclave
  • Stirring plate and magnetic stirrer
Troubleshooting
Before start
This protocol creates 1L of media.
Creating the Broth
Put a stir bar into a 1L glass bottle and fill the glass bottle with 1L of DI water (using a graduated cylinder). Mark the level of the water with a thin Sharpie or label tape. Pour out ~50mL of the water.
Note
We do this step because QSing the media in a graduated cylinder gets messy - it's often difficult to transfer the solute back and forth.

2m
In a large glass bottle (at least 1L), add the following:

Component Amount
Deoinized Water Amount950 mL
Tryptone Amount10 g
Yeast Extract Amount5 g
NaCl Amount10 g
(You do not have to add the exact amount of water, just make sure it is close to 950mL).
10m
Shake or stir to dissolve all the solutes.
20m
Optional: We have never done this step, but it is recommended by the Sambrook Molecular Cloning manual.

Adjust the pH of the solution to 7.0 using 5M NaOH (~0.2mL).
10m
QS the solution to the mark you made in Step 1 (1L) using DI water.
1m
Sterlizing the Broth
Autoclave bottle follwing Autoclave protocol on the liquid cycle. Choose the appropriate cycle for the amount of liquid you have. Make sure the bottle cap is on loosely
1h
Let bottle cool on the lab bench with loose cap
30m
Keep bottle in the cold room for storage, with the cap tightly closed. Make sure you keep the bottle STERILE. We want a clear solution and nothing growing in our broth