Mar 06, 2023
Preparation and transformation of electrocompetent cells
This protocol is a draft, published without a DOI.
- 1University of Oslo
Protocol Citation: Andreas Sagen 2023. Preparation and transformation of electrocompetent cells. protocols.io https://protocols.io/view/preparation-and-transformation-of-electrocompetent-cp26vqhe
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: February 26, 2023
Last Modified: March 06, 2023
Protocol Integer ID: 77630
Keywords: bacteria, electrocompetent, GYT medium
Abstract
A protocol for electroporation of E. coli. Other bacteria may work, with optimization of transformation buffer and settings.
Materials
Centrifuge
LAF
Vortex
Incubator
Electroporator
Protocol materials
TryptoneMerck Millipore (EMD Millipore)Catalog #T9410
Step 2
GlycerolMP BiomedicalsCatalog #194680
Step 2
Select Yeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
Step 2
Preparation of GYT medium
Preparation of GYT medium
In a sterile flask, add 400 mL distilled water
Measure and add 0.6 g yeast extract, 1.25 g tryptone, and 50 mL glycerol
Materials:
Select Yeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
TryptoneMerck Millipore (EMD Millipore)Catalog #T9410
GlycerolMP BiomedicalsCatalog #194680
Filter sterilize through a 0.22-µm filter, and store in aliquots at 4 °C
Preparation of cells
Preparation of cells
Inoculate 500 mL of prewarmed LB medium from 25 mL overnight E. coli culture. Incubate at 37 °C and 300 rpm. Measure OD every 20 minutes, until OD600=0.4
Note
The density is usually archived after ~2.5 hours of incubation with DH5α
Transfer the culture to appropriate centrifugation containers, and cool on ice for 00:30:00
30m
Centrifuge culture at 1000 rcf, 4°C, 00:15:00 , and resuspend pellet in 200 mL ice-cold water
Note
Combine the culture into a smaller number of centrifugation containers
15m
Centrifuge culture at 1000 rcf, 4°C, 00:20:00 , and resuspend pellet in 100 mL ice-cold 10% glycerol
20m
Centrifuge culture at 1000 rcf, 4°C, 00:20:00 , and resuspend pellet in 25 mL ice-cold 10% glycerol
20m
Centrifuge culture at 1000 rcf, 4°C, 00:20:00 , and resuspend pellet in 2 mL ice-cold GYT medium
Note
This is best done by gentle swirling rather than pipetting or vortexing
20m
Measure OD600 of a 1:100 dilution of the cell suspension, and dilute the cells to between 2-3 × 1010 cells/mL
Note
1.0 OD600=~2.5 X 108 cells/ml
Transfer 40 µl of the suspension to an ice-chilled electroporation cuvette and test whether arching occurs when an electrical discharge is applied. If so, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low
Dispense 40 µL aliquots of the cell suspension into sterile, ice-cold microfuge , drop into a bath of liquid nitrogen, and transfer to a -80 °C freezer
Transformation
Transformation
1h 5m
1h 5m
Pre-chill cuvettes on ice for 00:05:00 , and pre-heat LB plates with an appropriate selection agent and SOC medium at 37 °C for 01:00:00
1h 5m
Mix an appropriate amount plasmid to an aliquot of electrocompetent cells and transfer to a pre-chilled cuvette
Note
100 pg pUC19 plasmid is appropriate in most cases, but a specific amount plasmid depend on many different factors and have to be optimized
Protocol
NAME
S. O. C. mediumCREATED BY
Andreas Sagen
Incubate plasmid-suspension mix for 1 minute, then perform electroporation with optimized settings
Flush 1 mL pre-heated S. O. C. medium, then transfer to a culture tube and recover at 37 °C and 200 rpm shaking for 01:00:00
1h
Plate an appropriate amount culture on selection agar plates