Preparing Chemically Competent Cells using the CaCl2/Glycerol Method

< Return to Transformation Protocol

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

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.

  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 to an OD600 of 0.05 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.

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

< Return to Transformation Protocol

-- Main.MattMcGuffie - 07 Jun 2018

Edit | Attach | Watch | Print version | History: r4 < r3 < r2 < r1 | Backlinks | Raw View | More topic actions

 Barrick Lab  >  ProtocolList  >  ProtocolsChemCompCellsCaCl2Glycerol

Contributors to this topic Edit topic KateElston, MattMcGuffie
Topic revision: r4 - 2021-09-17 - 20:59:27 - Main.KateElston
 
This site is powered by the TWiki collaboration platform Powered by Perl This site is powered by the TWiki collaboration platformCopyright ©2024 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback