Nov 14, 2023

Public workspaceCportucalensisElectrocompetentCells

DOI
dx.doi.org/10.17504/protocols.io.kqdg3x7r7g25/v1
  • 1Stanford University;
  • 2California Institute of Technology;
  • 3UT Southwestern
Lev Tsypin
Open access
DOI: dx.doi.org/10.17504/protocols.io.kqdg3x7r7g25/v1
Protocol Citation: Lev Tsypin, Dianne K Newman, Allen W Chen, Scott Saunders 2023. CportucalensisElectrocompetentCells. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3x7r7g25/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: November 04, 2023
Last Modified: November 14, 2023
Protocol Integer ID: 90420
Keywords: Citrobacter portucalensis, electroporation, electrocompetent
Funders Acknowledgement:
NIH
Grant ID: 1R01AI127850-01A1
Doren Family Foundation
NSF GRFP
Abstract
Protocol to prepare electrocompetent Citrobacter portucalensis MBL cells.
Large batch electrocompetent cells
Large batch electrocompetent cells
Prepare 100 mL sterile LB in a 500 mL Erlenmeyer flask.

Prepare ice-cold 10 % glycerol and ultrapure water (both sterile).
Two days prior to electroporation, streak out strain on LB agar and grow at 30 ºC.
One day prior to electroporation, inoculate a 5 mL LB liquid culture with a patch of cells from the overnight streak and incubate slanted, shaking at 250 rpm at 30 ºC overnight.
Day of the electroporation, inoculate 1 mL of the overnight culture into the 100 mL LB flask and grow to OD600 = 0.4, shaking at 250 rpm at 30 ºC.
Chill flask on ice for 20 minutes.
Wash two the culture two times into ice-cold water (5000 x g for 10 minutes at 4 ºC; I typically divide the 100 mL volume into four 50 mL conical tubes, washing with 25 mL volumes).
Combine pellets and spin final time.
Resuspend in 2 mL ice-cold 10% glycerol.
Aliquot 50 µL volumes into ice-cold microcentrifuge tubes and flash-freeze in liquid nitrogen.
Store at -80 ºC or use immediately.
Small batch electrocompetent cells
Small batch electrocompetent cells
Two days prior, streak out strain as above.
One day prior, grow 5 mL LB culture overnight as above.
Day of, inoculate 50 µL overnight culture into 5 mL LB and incubate slanted, shaking at 250 rpm at 30 ºC until OD600 = 0.3.
Chill on ice for 10 minutes.
Wash three times into ice-cold water, final resuspension of combined pellets 50 µL (can also spin full 5 mL culture in conical tube directly).