Oct 18, 2019
Making LB agar plates
- 1Imperial College London
- Behavioural Genomics
Protocol Citation: Priota Islam 2019. Making LB agar plates. protocols.io https://dx.doi.org/10.17504/protocols.io.8eqhtdw
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: October 18, 2019
Last Modified: October 18, 2019
Protocol Integer ID: 28848
Abstract
Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. The addition of agar to LB results in the formation of a gel that bacteria can grow on, as they are unable to digest the agar but can gather nutrition from the LB within. When required the addition of an antibiotic to this gel allows for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid carrying the antibiotic resistance gene.
See the link for more details: https://www.addgene.org/protocols/pouring-lb-agar-plates/
Purchase LB Agar from the media kitchen and autoclave it to melt
Content of LB Agar:
37 g pre-mixed powder consisting of:
- 5 g peptone
- 10 g peptone from casein
- 10 g sodium chloride
- 10 g g agar-agar
1 L Sterile H2O
If the bacteria have a specific antibiotic resistance then add 1000x of that antibiotic to the melted LB agar before pouring, e.g. if we use ampicillin we add 100mg/ml to the LB in a way that the final concentration becomes 100ug/ml. So, for 200ml LB agar we add 200ul of 100mg/ul of stock ampicillin
| Antibiotic | Recommended Stock Concentration | Recommended Working Concentration | |
| Ampicillin | 100 mg/mL | 100 µg/mL | |
| Bleocin | 5 mg/mL | 5 µg/mL | |
| Carbenicillin | 100 mg/mL | 100 µg/mL | |
| Chloramphenicol | 25 mg/mL(dissolve in EtOH) | 25 µg/mL | |
| Coumermycin | 25 mg/mL(dissolve in DMSO) | 25 µg/mL | |
| Gentamycin | 10 mg/mL | 10 µg/mL | |
| Kanamycin | 50 mg/mL | 50 µg/mL | |
| Spectinomycin | 50 mg/mL | 50 µg/mL | |
| Tetracycline | 10 mg/mL | 10 µg/mL |
Antibiotic Concentrations
Maintaining sterile techniques, pour the molten agar on large petri dishes (90mm) roughly about 30-45ml (don’t need to be exact on the volume) and leave to dry overnight
Store the plates at 4C to be used later, ideally it should be used within 2 weeks but can be used for longer if not contaminated or dehydrated but never more than 3months