Protocol Citation: Ken Christensen 2020. Electroporation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkpukvnw
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: September 02, 2020
Last Modified: September 02, 2020
Protocol Integer ID: 41428
Abstract
This protocol may be used with electrocompetent cells prepared by you according to this protocol.
Guidelines
Appropriate Antibiotics for Your Application
Antibiotics for Plasmid selection
| Antibiotic | Working Concentration | |
| Ampicillin | 100 µg/ml | |
| Carbenicillin | 100 µg/ml | |
| Chloramphenicol | 33 µg/ml | |
| Kanamycin | 30 µg/ml | |
| Streptomycin | 25 µg/ml | |
| Tetracycline | 15 µg/ml |
Electroporation Protocol
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
Calculation:
If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)
cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)
Safety warnings
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.
Before start
For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF
Place recovery SOC in 37°C water bath
Pre-warm LB-antibiotic plates at 37°C
Thaw cells on ice for 10 min or use freshly made cells
00:10:00
Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice
Flick the tube containing cells a few times to mix and add 25 µl of competent cells to the microcentrifuge tube
25 µL
Add 1 µl of a 100 pg/µl to 1 ng/µl DNA solution (in DI water) to the cells in the microcentrifuge tube
1 µL
Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (you don’t hold the button down)
Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a microcentrifuge tube, 5 ml culture tube, or 15 ml centrifuge tube.
975 µL
Place in the shaker/incubator at 37°C incubator for 1 h
01:00:00
Make appropriate dilutions
Note
When using 100 pg - 1 ng of DNA, make three dilutions:
Dilute 1 µl of cells into 990 µl SOC and plate 100 µl. (10000-fold dilution)
Dilute 10 µl of cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
Dilute 100 µl of cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
Incubate overnight at 37°C
16:00:00