Griffitts:Chemically competent cells

Procedure
Note: After this point the cells should never touch anything that is warm&mdash;chill solutions, pipets, tubes, etc. beforehand
 * 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
 * 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

Mg2+/Ca2+
Autoclave
 * 3.25 g MgCl2·6H2O
 * 0.6 g CaCl2·2H2O
 * 200 mL dH2O

100 mM Calcium Chloride
Autoclave Note: You can also make a 1:10 dilution of the 1 M stock
 * 2.95 g CaCl2·2H2O
 * 200 mL dH2O