Oct 05, 2025
Electroporation
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnrneyl5d/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: 228975
Keywords: plasmid into dh10b, designed plasmid, plasmid, electroporation, dh10b, supporting orthogonal replication, orthogonal replication
Abstract
Introduce the designed plasmid into DH10B to establish E. coli supporting orthogonal replication.
Materials
- Plasmid
- Pre-prepared DH10B competent cells
- SOB medium pre-warmed to 37 °C
- Sterile electroporation cuvette (2 mm gap)
- Antibiotic agar plates (according to the resistance gene carried by the plasmid)
- Electroporator
- Shaking incubator at 37 °C (220 rpm)
- Biosafety cabinet
- Ice bucket with ice
Troubleshooting
Preparation of the Mixture
Take out pre-prepared DH10B competent cells and thaw on ice (~10 minutes).
In a sterile workspace, add 1.0 μL of plasmid DNA (dissolved in ultrapure water, ~50–100 ng/μL) to the thawed competent cells. Do not stir, pipette harshly, or create bubbles.
Carefully transfer the mixture into a pre-chilled 2 mm electroporation cuvette (kept on ice ≥10 minutes). Close the lid and leave on ice for 2 minutes.
Electroporation
Place the cuvette into the electroporator and set the following conditions:
- Voltage: 2500 V (standard for 2 mm gap)
- Time constant: 4.5–5.5 ms (ideal range)
- Pulse number: 1 pulse, duration should not exceed 6 ms
A faint spark sound should be heard during electroporation. If the instrument displays “Arcing” (discharge failure), replace the cuvette immediately and check whether the sample contains salt.
Immediate Recovery
Immediately add 1 mL of pre-warmed (37 °C) LB medium into the cuvette and gently pipette to mix.
Transfer the cuvette contents into a sterile 15 mL culture tube.
Incubate at 37 °C in a shaking incubator at 220 rpm for 60 minutes (recovery culture).
Antibiotic Selection (see detailed steps in next protocol)
After 1 hour of recovery, plate 100 μL of the culture onto an LB agar plate containing the appropriate antibiotic.
Incubate the plate at 37 °C in an inverted position (agar side up) overnight (12–16 hours).