Jan 25, 2025
Electroporation & Electrocompetent Cell Preparation Protocol for DAP auxotroph E.coli WM3064
Forked from Electroporation Protocol
- 1New England Biolabs;
- 2The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway, Tromsø, Norway;
- 3Microalgae & Microbiomes Research Group (M2RG), UiT - The Arctic University of Norway, Tromsø, Norway;
- 4The Arctic Centre for Sustainable Energy (ARC), UiT - The Arctic University of Norway, Tromsø, Norway
Protocol Citation: New England Biolabs, Atharva Karde, Hans Christopher Bernstein 2025. Electroporation & Electrocompetent Cell Preparation Protocol for DAP auxotroph E.coli WM3064. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1rxwlmk/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 regularly use this protocol.
Created: October 02, 2024
Last Modified: January 25, 2025
Protocol Integer ID: 108862
Keywords: Electroporation, Competent cells, DAP auxotroph, WM3064, Transformation, Electrocompetent, Ecoli, E.coli,
Funders Acknowledgements:
Kometen – Nord-Norges forskningsfond
Abstract
This is a protocol for making electrocompetent E.coli WM3064 cells and transforming them with DNA through electroporation. This protocol produces 30 µL aliquots of electrocompetent cells, each enough for one prep of electroporation.
Guidelines
Electroporation Protocol
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.
Transformation efficiency 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 plasmid (e.g. 10 pg per ml)
Transformation efficiency = cfu/ µg = (colonies counted*1000) / (0.00001 µg plasmid)
Materials
MATERIALS
Magnesium sulfate heptahydrateSigma AldrichCatalog #M2773
NaClSigma AldrichCatalog #53014
TryptoneFisher ScientificCatalog #BP1421-500
GlucoseSigma AldrichCatalog #G8270
Magnesium chloride hexahydrateSigma AldrichCatalog #M2670
Potassium chlorideSigma AldrichCatalog #P9333
GlycerolThermo Fisher ScientificCatalog #17904
Yeast ExtractThermo FisherCatalog #211930
2,6-Diaminopimelic acidThermo Scientific
Media
SOB + DAP:
2% tryptone
0.5% yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
0.3 mM DAP
SOC + DAP :
SOB + 20 mM glucose + 0.3 mM DAP
LB agar + DAP + antibiotic
LB agar + 0.3 mM DAP + appropriate antibiotic
Appropriate Antibiotics for Your Application
Antibiotics for Plasmid selection
| A | B | |
| Antibiotic | Working Concentration | |
| Ampicillin | 100 µg/ml | |
| Carbenicillin | 100 µg/ml | |
| Chloramphenicol | 33 µg/ml | |
| Kanamycin | 50 µg/ml | |
| Streptomycin | 25 µg/ml | |
| Tetracycline | 15 µg/ml |
Protocol materials
2,6-Diaminopimelic acidThermo Scientific
Materials, Step 3
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Materials
Yeast ExtractThermo FisherCatalog #211930
Materials
Magnesium sulfate heptahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2773
Materials
GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8270
Materials
Magnesium chloride hexahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M2670
Materials
GlycerolThermo Fisher ScientificCatalog #17904
In Materials and 2 steps
TryptoneFisher ScientificCatalog #BP1421-500
Materials
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
Materials
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Quite a bit of glycerol gets left behind in the tube during the washing steps while discarding the supernatant. Note that this dilutes the cells a bit. Prepare aliquots based on the cell quantity required specifically for your use.
Dilute the plasmid to have 1 - 3 µL of DNA (<100 ng) per electroporation prep. Calculate the amount of DNA used per prep to determine the transformation efficiency later.
The electroporation mixture ratio will vary depending on the strain and DNA size so this protocol may need to be optimized.
Make sure to include a negative control which also serves as a "survival control". This mixture should contain only electrocompetent cells and no plasmid. Plate this mixture on LB+DAP agar to assess whether the cells survive the electroporation pulse.
This protocol was developed as part of a master's thesis affiliated with the Microalgae & Microbiomes Research Group at NFH, UiT, Norway.
M2RG Website : https://uit.no/research/micro
Growing overnight culture
Growing overnight culture
Add a loopful of bacterial culture to 5 mL LB broth.
Incubate Overnight at 37 °C with shaking.
Making electrocompetent cells
Making electrocompetent cells
Add 10 mL SOB medium containing 0.3 millimolar (mM) 2,6-Diaminopimelic acidThermo Scientific in a 50 mL conical flask.
Inoculate each flask with overnight culture to OD600 = 0.05
Grow cells until mid-exponential phase (OD600 = 0.6) or 02:00:00
Transfer the cells to a 15 mL Falcon tube
Chill the culture On ice for 00:10:00 .
Centrifuge the cells at 6000 rpm, 4°C, 00:05:00 .
Discard the supernatant.
Wash the pellet by adding 10 mL chilled 10 % volume GlycerolThermo Fisher ScientificCatalog #17904 and vortex vigourously.
Centrifuge 6000 rpm, 4°C, 00:03:30 and discard the supernatant.
go to step #10 Repeat for a total of 4 wash cycles .
Resuspend the washed pellet in 100 µL 10 % volume GlycerolThermo Fisher ScientificCatalog #17904
Make 30 µL aliquots in sterile microcentrifuge tubes. Proceed to electroporation or store at -80 °C . One tube allows for one prep of electroporation.
Thawing and preparation
Thawing and preparation
Turn on electroporator and set to 1.5 kv, 200 ohms and 25 µF.
Pre-warm recovery SOC+DAP medium and LB-antibiotic plates at 37 °C .
Thaw microcentrifuge tubes with electrocompetent cells On ice or use freshly made cells.
Place appropriate number of 1 mm-electroporation cuvettes On ice .
Electroporation and recovery
Electroporation and recovery
Add 1 µL DNA solution (<100 ng) to 30 µL electrocompetent cells in microcentrifuge tubes.
Note
This mixture can be optimized for your strain and plasmid. Trying a few combination ratios is recommended in order to determine which one yields the highest transformation efficiency. A lower amount of DNA usually yields higher efficiency.
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 970 µL 37°C SOC + DAP , mix by pipetting up and down once and transfer to a microcentrifuge tube.
Note
Try to add the recovery medium as soon as possible. Transformation efficiency sharply declines with increase in time between pulse and addition of recovery medium.
Incubate at 37 °C for 01:00:00 .
Dilution plating and efficiency calculation
Dilution plating and efficiency calculation
Make appropriate dilutions and plate 100 µL on LB agar + DAP + antibiotic plates.
Incubate Overnight at 37 °C .
Note
Transformation efficiency 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 plasmid (e.g. 10 pg per ml)
Transformation efficiency = cfu/ µg = (colonies counted*1000) / (0.00001 µg plasmid)
Protocol references
- Short Protocols in Molecular Biology, Chapter 1.
- Datsenko, K.A., and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97: 6640-6645.
Acknowledgements
This protocol was developed as part of a master's thesis affiliated with the Microalgae & Microbiomes Research Group at NFH, UiT, Norway.
M2RG Website : https://uit.no/research/micro
The E.coli strain used in this project was generously provided by the Lowe-Power Lab at UC Davis (https://lowepowerlab.ucdavis.edu/).