Oct 05, 2025

Public workspaceE.coli DH10B Competent Cell Preparation

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx415dl8j/v1
  • igemnthuth 1
  • 1iGEM NTHU_Taiwan
  • iGEM NTHU
igemnthuth
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx415dl8j/v1
Protocol Citation: igemnthuth 2025. E.coli DH10B Competent Cell Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx415dl8j/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: 228956
Keywords: competent cells suitable for electroporation, subsequent plasmid introduction, hosts for subsequent plasmid introduction, electroporation, plasmid, competent cell, cell, preparation
Abstract
Preparation of E. coli competent cells suitable for electroporation, to serve as hosts for subsequent plasmid introduction.
Materials
  • Cultured log-phase DH10B cells (200 mL culture, OD600 ≈ 0.6–0.8)
  • Ice-cold sterile 10% glycerol solution (filter-sterilized, pre-chilled at 4 °C or on ice, prepare at least 100 mL)
  • Sterile centrifuge bottles (250–500 mL)
  • Sterile microcentrifuge tubes (1.5 mL, 10 tubes)
  • Sterile pipette tips and micropipettes
  • Ice bucket and crushed ice bath (all procedures must be kept cold)
  • Refrigerated centrifuge (set to 4 °C, 4500 × g)
  • Shaking incubator (37 °C, 220 rpm)
Troubleshooting
E.coli DH10B Competent Cell Preparation
1h 10m
Obtain log-phase culture (OD600 ≈ 0.6–0.8) and immediately place it on ice to cool for 10 minutes (gently swirling during cooling is recommended).
10m
Transfer 200 mL of culture into pre-chilled centrifuge bottles and centrifuge at 4500 × g for 10 minutes at 4 °C.
10m
Carefully discard the supernatant without disturbing the cell pellet.
5m
Add 50 mL of ice-cold 10% glycerol. Mix by repeatedly pipetting up and down until the suspension is evenly turbid. Centrifuge again at 4500 × g for 10 minutes at 4 °C.
10m
Repeat steps 3–4 for a total of two washes.

30m
After the final centrifugation, discard the supernatant and resuspend the entire pellet in 1 mL of ice-cold 10% glycerol. Transfer the suspension into sterile 1.5 mL tubes.
5m
Aliquot 50–100 μL per tube. Keep on ice for immediate use, or snap-freeze in liquid nitrogen and store at −80 °C (stable for 1–2 weeks).