Oct 05, 2025

Public workspaceAntibiotic Selection

DOI
dx.doi.org/10.17504/protocols.io.q26g7nwdklwz/v1
  • igemnthuth 1
  • 1iGEM_NTHU_Taiwan
  • iGEM NTHU
igemnthuth
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7nwdklwz/v1
Protocol Citation: igemnthuth 2025. Antibiotic Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7nwdklwz/v1
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 03, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228979
Keywords: select colonies with the antibiotic resistance marker, antibiotic selection, synthetic ecorep operon, antibiotic resistance marker, plasmid, dh10b
Abstract
Confirm that E. coli DH10B has successfully taken up the plasmid carrying the synthetic EcOrep operon, and select colonies with the antibiotic resistance marker via antibiotic selection.
Materials
  • Recovered culture (per tube: 100 µL competent cells + 6 µL plasmid + 1 mL SOB medium; recover at 37 °C for 2 h)
  • LB agar powder (containing 1% tryptone, 0.5% yeast extract, 1% NaCl, 2% agar)
  • [Antibiotic] stock solution: 20 mg/mL, dissolved in 100% ethanol, stored at −20 °C
  • ddH₂O
  • LB agar plates: final [Antibiotic] concentration 20 µg/mL per plate (see preparation steps below)
  • Sterile 1.5 mL Eppendorf tubes
  • 100 mm sterile Petri dishes
  • Sterile glass spreader
  • Micropipettes (20 µL, 200 µL, 1000 µL)
  • 37 °C incubator
Troubleshooting
Preparation of Antibiotic Agar Plates
Weigh 25 g LB agar powder using an electronic balance and dissolve in 1 L ddH₂O.
Autoclave at 121 °C, 15 psi, for 1–2 hours.
After autoclaving, cool the medium to ~50 °C (check with an infrared thermometer).
Add 1 mL of 20 mg/mL [Antibiotic] stock to 1 L of medium (final concentration: 20 µg/mL) and mix thoroughly. (Ensure the medium has cooled sufficiently before adding antibiotic.)
Pour 25 mL of the medium into each sterile Petri dish.
Allow to solidify at room temperature. Seal with Parafilm, label with date and antibiotic name, and store at 4 °C (up to 7 days).
Dilution and Plating of Recovered Cells
Mix the recovered culture thoroughly, then split into two groups:

  • Undiluted group: Take 100 µL of undiluted culture.
  • 1:10 dilution group: Mix 100 µL culture with 900 µL SOB; from this, take 100 µL.
Spread each 100 µL sample evenly onto LB agar plates containing the antibiotic.
Use a sterile glass spreader to distribute the liquid.
Let the surface dry (~5 minutes), then invert the plates.
Incubation and Observation
Place the plates in a 37 °C incubator, inverted, and incubate for 14–16 hours.
On the following day, observe colony formation:

  • Presence of colonies indicates successful transformation.
  • If colonies are too dense on the undiluted plate, use colonies from the 1:10 dilution plate instead (adjust depending on culture density).