Griffitts:Chemically competent cells

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