Pelling:Protocols/Preparing and Transforming Chemically Competent Bacteria

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Contents

Preparing DH5α Chemically Competent Cells

Required Materials

  • 2M CaCl2 Stock Solution (22.198 grams CaCl2, Anhydrous into 100mL sterile dH2O and filter sterilize).
  • LB Medium
  • LB-Agar Plates (no antibiotics)
  • Ice cold dH2O
  • Autoclave 6 x 50mL tubes
  • Autoclave 500mL Erlenmeyer Flask
  • Autoclave 15 x 1.5mL microcentrifuge tubes

CaCl2 Competent Cell Stock Protocol

  • Day 1 Afternoon: Streak cells from a frozen stock and grow overnight.
  • Day 2 Afternoon: Pick single colony and grow in 5mL LB media overnight (300rpm).
  • Day 3 Morning:
    • Chill the centrifuge to 4°C and continue to Steps 2 and 3. DO NOT start Step 4 before centrifuge reaches 4°C.
    • Make 20mL of 0.5M CaCl2 in ice cold sterile dH2O in a sterile 50mL tube. Leave on ice in the fridge. (5mL 2M CaCl2 + 15mL dH2O).
    • Make 6mL sterile freezing media (0.1M CaCl2 / 15% Glycerol) in a sterile 50mL tube. Leave on ice in the fridge. (0.3mL 2M CaCl2 + 0.9mL 100% Glycerol + 4.8mL dH2O).
    • Transfer into a 500mL Erlenmeyer Flask with 195mL LB media and grow cells until an OD of 0.5-0.6 A600 is achieved.
    • Split cells into 4 x 50mL sterile tubes and put on ice and keep chilled at 0°C for 15min.
    • Keep everything from this point onwards at 4°C or lower.
    • Spin cells at 4000rpm for 10 min at 4°C.
    • Remove supernatant, collect and re-suspend cells with 15mL ice-cold 0.5M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave cells on ice for 15min.
    • Spin again at 4000rpm for 10 min at 4°C.
    • Re-suspend in 6mL sterile, ice-cold 0.1M CaCl2 /15% glycerol (DO NOT VORTEX).
    • Optional: Leave on ice 4 to 21 hours (DH5α can be left overnight).
    • Freeze 500μL aliquots of cells in sterile and labeled microcentrifuge tubes.

Transforming DH5α Chemically Competent Cells

  1. Thaw an aliquot of chemically competent bacteria on ice for 15min.
  2. Add 1-2μl (up to 10μl) of plasmid DNA (ng amounts) to 50μl of bacteria in a sterile tube. Want to use small amounts of DNA as large volumes will dilute bacteria buffer and reduce efficiency. Keep previously extracted DNA samples in case anything goes wrong.
  3. Incubate tubes on ice for 30min.
  4. Heat shock cells by placing tube in water at 42°C for exactly 45sec.
  5. Immediately transfer tubes to ice for 2min.
  6. Add cold 250μl of LB or SOC media. SOC better if LB doesn’t work.
  7. Cap the tube tightly and put the tubes horizontally in the shaker (tape them down) and shake at 37°C for 60-90mins.
  8. If necessary make dilutions of the bacteria in (1:10, 1:2 or 1:1) in LB in new tubes.
  9. Place 100μl of bacteria in center of an appropriate LB plate and spread evenly over whole surface with bent Pasteur pipette or any other loop, tip or spreader.
  10. Most strains require 12-18 hours to form colonies. Do not incubate for excessive times as satellite colonies will form (E.coli form small colonies). Plates/colonies can be stored for up to a week (with parafilm) at 4°C if not to be used immediately.
  11. Place any unused chemically competent bacteria back in the -80°C freezer and mark top with a black 'X' to denote that it has been thawed at least once.
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