Oct 23, 2025
BL-21 Competency CaCl2 Treatment
- mpalacios 1
- 1Liberty University
Protocol Citation: mpalacios 2025. BL-21 Competency CaCl2 Treatment. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3119el25/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: October 22, 2025
Last Modified: October 23, 2025
Protocol Integer ID: 230520
Keywords: Competency, competency cacl2 treatment protocol for calcium chloride treatment, competency cacl2 treatment protocol, calcium chloride treatment, competency
Abstract
Protocol for Calcium Chloride treatment to induce competency in BL-21 cells.
Materials
PER COLONY -
55 1.5mL sterile microcentrifuge tubes
5mL of sterile 100mM CaCl2
2mL of sterile 100mM CaCl2 + Glycerol
1 plate of non-selective media (LB Agar or TSA)
60mL of sterile LB (10mL in a falcon, 50mL in a 250mL shaking flask)
Microcentrifuge with temperature control (pre-chilled to 0ºC)
Cold room is helpful
Ice Water bath
Safety warnings
Due to working in non-selective media, ensure to follow sterile protocol at all steps.
Cell Growth
4h 15m
Plate your BL-21 on non-selective plates (LB Agar or TSA)
20m
Incubate overnight at 37 °C
Using a sterile pipette tip, pick a colony and add to 10mL of non-selective LB
20m
Incubate overnight at 140 rpm, 37°C, 12:00:00
Gather 55 sterile 1.5mL tubes and add to freezer to chill
Consider labeling the tubes at this point to save time later
Add 500 µL of overnight culture to 50 mL of non-selective LB
5m
Incubate 210 rpm, 37°C, 02:00:00
2h
You can also label tubes now.
Measure OD600 every 00:30:00 until early to mid-log phase (0.35-0.55)
1h 30m
Take colony and add to wet ice to halt growth.
Everything from this point forward needs to be as close to 0ºC as possible.
CaCl2 Treatment
3h 50m
Gather 5 prechilled 1.5mL tubes
Add 1mL of culture to each tube
Centrifuge the tubes at8000 rpm, 0°C, 00:05:00
5m
Discard supernatant
Repeat steps 11-13 9 times until a total of 10mL worth of cells is added to each tube
1h
Resuspend each tube in 1mL of 100 millimolar (mM) CaCl2 by pipetting
10m
Incubate on ice for 02:30:00
2h 30m
Centrifuge the tubes at 8000 rpm, 0°C, 00:05:00
5m
Discard supernatant and resuspend pellet in 500mL of 100 millimolar (mM) CaCl2 + 15 % volume glycerol
Last time pipetting up and down is possible, from here on out mix by flicking
Aliquot 50 µL into pre-chilled tubes.