Griffitts:Chemically competent cells

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Contents

Procedure

  • Grow DH5α (B095) overnight in 5 mL LB at 37°C
  • Inoculate 100 mL LB with 1 mL of saturated overnight culture of DH5α
  • Shake at 37°C until OD600 = 0.4
    • Usually 2–3 hours
  • Place in an ice bath for 5 minutes

Note: After this point the cells should never touch anything that is warm—chill solutions, pipets, tubes, etc. beforehand

  • Divide the culture into 2 tubes with ~40 mL each
  • Centrifuge at 8000 rpm for 10 minutes in a cold SS34 rotor
    • The Sorvall Centrifuge is found in the Harker/Breakwell lab (747 WIDB)
  • Gently resuspend each pellet with 15 mL of cold Mg2+/Ca2+ solution
  • Incubate in an ice bath for 30 minutes
  • Centrifuge at 8000 rpm for 10 minutes
  • Resuspend each pellet with 1.6 mL of cold 100mM CaCl2 solution
  • Incubate in an ice bath for 20 minutes
  • Combine cells to one tube
  • Add 0.5 mL cold 80% glycerol and swirl to mix
  • Flash-freeze in liquid nitrogen as 100-μL aliquots
  • Store in the –80°C freezer

Transformation

  • Add 7 μL ligation mix to chemocompetent DH5α cells
  • Incubate on ice for 5–10 minutes
  • Heat shock at 42°C for 1 minute
  • Incubate on ice for 2 minutes
  • Add 800 μL LB
  • Tape to a shaker at the appropriate temperature for 1 hr.
  • Plate 50 μL and 250 μL onto the appropriate selective medium

Solutions

Mg2+/Ca2+

  • 3.25 g MgCl2·6H2O
  • 0.6 g CaCl2·2H2O
  • 200 mL dH2O

Autoclave

100 mM Calcium Chloride

  • 2.95 g CaCl2·2H2O
  • 200 mL dH2O

Autoclave
Note: You can also make a 1:10 dilution of the 1 M stock

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