Feb 14, 2022
Version 2
Electroporation Protocol V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2022. Electroporation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bd22i8geVersion created by New England Biolabs
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: March 22, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 34618
Keywords: Electroporation, Competent cells,
Abstract
This protocol may be used with electrocompetent cells prepared according to this protocol.
Guidelines
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)
Materials
MATERIALS
Magnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2773
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
TryptoneFisher ScientificCatalog #BP1421-500
GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270
Magnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2670
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
GlycerolThermo Fisher ScientificCatalog #17904
Yeast ExtractThermo FisherCatalog #211930
Media
SOB:
2% tryptone
0.5% yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
SOC:
SOB + 20 mM glucose
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 |
Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
For control electroporation dilute pUC19 to 10 pg/μl with Milli-Q water.
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.
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 00:10:00 or use freshly made cells.
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 to the microcentrifuge tubes.
Add 1 µL 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
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 (don’t hold the button down).
Immediately add 975 µL 37°C SOC , mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
Rotate in the 37 °C incubator for 01:00:00 .
Make appropriate dilutions.
Note
When using 10 pg DNA , make two dilutions:
Dilute 10 µL cells into 990 µL SOC and plate 100 µL . (1000-fold dilution)
Dilute 100 µL cells into 900 µL SOC and plate 100 µL . (100-fold dilution)
Incubate Overnight at 37 °C .
Note
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)