Jan 14, 2025

Public workspaceLuria Delbruck– Ampicillin Plates

This protocol is a draft, published without a DOI.
  • 1UC Irvine
Jaycee Fahrner
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Protocol Citation: Jaycee Fahrner, Maria Rebolleda-Gomez 2025. Luria Delbruck– Ampicillin Plates . protocols.io https://protocols.io/view/luria-delbruck-ampicillin-plates-dt996r96
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: December 10, 2024
Last Modified: January 14, 2025
Protocol Integer ID: 114721
Keywords: Luria-Delbruck, Antibiotics, Selection
Disclaimer
Adapted protocol from Maria Rebolleda-Gomez. See attachment.
Abstract
Adapted from Luria Delbruck protocol Spring2024 for E115L Evolution Lab class. Many populations of E. coli are grown in nutrient rich media then diluted and plated on ampicillin as selective environment at the MIC of this strain ~2 ug/mL. Expect many plates without CFUs and few plates with successful colonization.
Attachments
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Materials
  • LB media
  • LB agar (2* 600 µL for 45 plates)
  • Ampicillin stock solution (100 mg/mL)
  • 45 Petri dishes
  • 96-well plate(s)
  • Plate sealing film
  • Microplate reader
  • Sterile glass beads


Day 1 – Grow overnight cultures and prepare antibiotic agar
Day 1 – Grow overnight cultures and prepare antibiotic agar
1d 0h 20m
1d 0h 20m
From a single colony, inoculate Amount2 mL of LB media and grow Duration24:00:00 at Temperature35 °C
1d
Incubation
Overnight
For 44 Concentration2 µg/µL ampicillin plates, prepare 2 bottles of Amount600 mL LB agar. Remove Amount12 µL water before adding to LB agar powder. Autoclave using liquid settings at Temperature121 °C with Duration00:15:00 sterilization time. Make 1 plate with normal LB agar (no antibiotics), prepare separate LB agar bottle. Place magnetic stir stick in bottles that will be used to make the antibiotic agar.

15m
After media is cool enough to touch the bottle with back of your hand without pain, add Amount12 µL of Concentration100 mg/mL ampicillin to each bottle. Use stir plate to mix for Duration00:05:00 .


Safety information
In working with antibiotics, use extra precaution and ensure proper PPE. After mixing in ampicillin, pour plates in a timely manner. Agar will solidify and magnetic stir bar cannot be microwaved/ antibiotics cannot be reheated.



5m
Mix
Critical
Toxic
Pour plates into sterile petri dishes. Allow to cool to room temp then store at Temperature4 °C

Day 2 – Portioning populations
Day 2 – Portioning populations
2d
2d
Using a plate reader, adjust OD620 to ~0.1 with LB
Dilute adjusted culture 10-3 using LB media
Plate Amount50 µL of diluted culture on LB agar (no antibiotics) using sterile glass beads. Incubate Duration24:00:00 at Temperature35 °C and record number of CFUs
Expected result
Shooting for 50-200 CFUs per Amount50 µL (such that there are 10-50 cells perAmount2 µL ). Adjust dilution factor as needed.



1d
Incubation
Overnight
Portion Amount198 µL LB media into first 4 rows of a 96-well plate

Inoculate Amount2 µL diluted culture into first four rows. Include a media control,
e.g. columns 1-11 inoculated (purple), A12 media control (blue):
12345
A
B
C
D
E
F
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H
678910
A
B
C
D
E
F
G
H
1112
A
LB only
B
C
D
E
F
G
H


Seal plate with clear plastic film and incubate Duration24:00:00 at Temperature35 °C

1d
Incubation
Overnight
Day 3 – Plating populations
Day 3 – Plating populations
1d
1d
In a new 96-well plate or in the bottom 4 rows of the original 96-well plate, portionAmount198 µL of LB media
Inoculate Amount2 µL from original populations into the fresh LB (red). Do the same for media control (blue).

12345
A
B
C
D
E
F
G
H
678910
A
B
C
D
E
F
G
H
1112
A
LB only
B
C
D
E
LB only
F
G
H


Plate Amount50 µL from each population (red wells) onto ampicillin agar plate. Shake with sterile glass beads. Do the same for media control (blue well).

Incubate inoculated agar plates for Duration24:00:00 at Temperature35 °C

1d