Preparing Chemically Competent Cells using the CaCl₂/Glycerol Method

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Adapted from: Re-engineering the ribosome for efficient selenoprotein synthesis Ross Thyer, 2012

http://docplayer.net/78150757-Re-engineering-the-ribosome-for-efficient-selenoprotein-synthesis.html

SUPPLIES:

Equipment:

Floor Centrifuge
Sterile 50mL Centrifuge Tubes
Shaking 37°C Incubator
Sterile 1L Flasks

Consumables:

Liquid Nitrogen
Polypropylene Microcentrifuge Tubes

Buffers and Solutions:

LB Medium

100 mM CaCl2 10% Glycerol Buffer:

Reagent	Amount / L	Final Conc.
CaCl₂	11.10 g	100 mM
80% Glycerol	125 mL	10%
H₂O	to 1 L

Filter sterilize and store at 4°C

PROTOCOL

The recipe below should yield 100 tubes of chemically competent cells. If fewer tubes are needed, volumes can be adjusted proportionally for steps 2-4.

) Competent stocks of E. coli strains were prepared by inoculating 10 ml of LB medium with a small portion of cells scraped from the surface of a frozen glycerol stock not previously exposed to CaCl2.
) Following incubation overnight at 37 °C with agitation, cultures were diluted to an OD600 of 0.05 in 500 ml LB medium and further incubated until reaching an OD600 between 0.4 and 0.6
) Aliquots of approximately 35 ml were centrifuged at 3400 g for 10 minutes at RT (Allegra X-22 Centrifuge, Beckman Coulter) and the combined pellets resuspended in 150 ml of ice cold 100 mM CaCl2/10% glycerol solution.
) Cells were centrifuged again at 3400 g for 10 minutes at RT (Allegra X-22 Centrifuge, Beckman Coulter) in aliquots of approximately 30 ml and the combined pellets resuspended in 20 ml ice cold 100 mM CaCl2/10% glycerol solution.
) Following incubation on ice for 25 minutes 200 µl aliquots of the cells were snap frozen in liquid nitrogen and stored at -80 °C.

**liquid nitrogen can be substituted with dry ice + 100% EtOH bath, or omitted entirely for slightly less competent cells