ProtocolsChemCompCellsCaCl2Glycerol < Lab

---+ Preparing Chemically Competent Cells using the CaCl<sub>2</sub>/Glycerol Method
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 Adapted from: <i>Re-engineering the ribosome for efficient selenoprotein synthesis</i> 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:

   * [[ProtocolsMediaRecipes#LB_Lysogeny_Broth_Luria_Bertani][LB Medium]]

    <b>100 mM CaCl2 10% Glycerol Buffer: </b>
| *Reagent* | *Amount / L* | *Final Conc.* |
| CaCl<sub>2</sub> | 11.10 g | 100 mM |
| 80% Glycerol |  125 mL  |  10% |
| H<sub>2</sub>O | to 1 L | |
   * 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
 

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-- Main.MattMcGuffie - 07 Jun 2018
This topic: Lab > WebHome > ProtocolList > ProtocolsChemCompCellsCaCl2Glycerol
Topic revision: r3 - 2020-04-09 - KateElston