Oct 04, 2022
CompetentCell
- Ana Belem García González1,
- Georgina Diego1,
- Irán Alessandra Chaparro Rodríguez1,
- Jair Alexis Gardea Sáenz1
- 1Tecnologico de Monterrey Campus Chihuahua
- Tec-Chihuahua_2022_Agrocapsi
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 05, 2022
Last Modified: October 04, 2022
Protocol Integer ID: 68197
Keywords: competent cell, competentcell, cell
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
CaCl
Competent cells protocol with CaCl2
1h 20m
Inoculate 100 µL of E. coli in10 mL of antibiotic-free starter LB culture medium. Incubate during Overnight at 37 °C and 225 rpm
Heat the LB media in the water bath at37 °C
Inoculate 40 mL of LB media with 1 mL of cell culture from step 1.
Incubate at 37 °C with constant agitation (225 rpm during 00:30:00 ).
30m
Measure de optical density at 600 nm every 00:30:00 until an OD600 of 0.4-0.6 is reached.
30m
Chill for00:20:00 On ice
20m
Centrifuge during 00:15:00 at 4 °C and 3000 rpm
15m
Discard the supernatant, add 20 mL of CaCl solution 50 mM and resuspend carefully.
Chill for 00:20:00 On ice
20m
Centrifuge during 00:15:00 at 4 °C and 3000 rpm
15m
Discard the supernatant, add 5 mL of CaCl2 solution 100 mM and resuspend carefully. ChillOn ice for 00:20:00
20m
Preserve at 4 °C