Oct 03, 2022

Public workspace CompetentCell

DOI
dx.doi.org/10.17504/protocols.io.j8nlkkzd6l5r/v1
  • 1Tecnologico de Monterrey Campus Chihuahua
Denisse Chacón Ramírez
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkkzd6l5r/v1
Protocol Citation: Ana Belem García González, Georgina Diego, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz 2022. CompetentCell. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkkzd6l5r/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: August 04, 2022
Last Modified: October 03, 2022
Protocol Integer ID: 68197
Abstract
A protocol for making chemically competent cells is shown.
Materials
  • LB Broth
  • CaCl2 50 mM Cold
  • CaCl2 100 mM Cold
  • 50 mL Falcon Tubes
  • E. coli cells

Equipment:
  • Spectrophotometer
  • Centrifuge
  • Incubator at 37 °C
  • Shaker
  • Water Bath at 37 °C
  • Cooler with ice
Protocol materials
ReagentCaCl
Step 8
Competent cells protocol with CaCl2
Competent cells protocol with CaCl2
1h 20m
1h 20m
Inoculate Amount100 µL of E. coli inAmount10 mL of antibiotic-free starter LB culture medium. Incubate during DurationOvernight at Temperature37 °C and Shaker225 rpm Centrifigation

Heat the LB media in the water bath atTemperature37 °C

Inoculate Amount40 mL of LB media with Amount1 mL of cell culture from step 1.

Incubate at Temperature37 °C with constant agitation (Shaker225 rpm during Duration00:30:00 ).

30m
Measure de optical density at 600 nm every Duration00:30:00 until an OD600 of 0.4-0.6 is reached.

30m
Chill forDuration00:20:00 TemperatureOn ice

20m
Centrifuge during Duration00:15:00 at Temperature4 °C and Centrifigation3000 rpm

15m
Discard the supernatant, add Amount20 mL of ReagentCaCl solution 50 mM and resuspend carefully.

Chill for Duration00:20:00 TemperatureOn ice

20m
Centrifuge during Duration00:15:00 at Temperature4 °C and Centrifigation3000 rpm

15m
Discard the supernatant, add 5 mL of CaCl2 solution 100 mM and resuspend carefully. ChillTemperatureOn ice for Duration00:20:00

20m
Preserve at Temperature4 °C