Preparing Chemically Competent Cells using the CaCl2/Glycerol Method
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:
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
- ) 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 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. 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
-- Main.MattMcGuffie - 07 Jun 2018