Preparing Chemically Competent Cells using the CaCl₂/Glycerol Method

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:

• Filter sterilize and store at 4°C

PROTOCOL

1. ) 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.
2. ) Following incubation overnight at 37 °C with agitation, cultures were diluted in 500 ml LB medium and further incubated until reaching an OD600 between 0.4 and 0.6
3. ) 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.
4. ) 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.
5. ) Following incubation on ice for 25 minutes 200 µl aliquots of the cells were snap frozen in liquid nitrogen and stored at -80 °C. Proportionally smaller volumes were used for steps following the overnight incubation when fewer stocks were required.

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