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 37C 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 4C

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 l 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.

-- Main.MattMcGuffie - 07 Jun 2018

 Barrick Lab  >  ProtocolList  >  ProtocolsChemCompCellsCaCl2Glycerol

Topic revision: r1 - 07 Jun 2018 - 20:00:38 - Main.MattMcGuffie
 
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