Competent cells

Preparation
  1. Pick one colony off fresh DH5() plate into 2.5 ml LB supplemented with 25 l 1M MgSO4 (10mM final conc.)
  2. Shake at 37°C overnight and until use
  3. Do a 1:500 dilution by inoculating 100 ml of SOB in 1 L flask with 200 l of o/n DH5(), record start time
  4. Spec starting at 3 hours after start time to an OD550 0.15 to 0.3 or "eye spec" every 20 minutes starting at 3 hours after start time.
  5. Collect in two pre-cooled 50 ml orange cap c/f tubes and incubate on ice 15 min
  6. Pellet in the hermle swinging bucket rotor at 2500 rpm for 5 min at 4°C with no brake, drain pellet thoroughly
  7. Resuspend in RF1 in 1/3 original volume(30 ml) by gently pipetting with DNA tips
  8. Pellet in the hermle swinging bucket rotor at 2500 rpm for 5 min at 4°C with no brake, drain pellet thoroughly
  9. Resuspend by gently swirling in RF2 in 1/12.5 original volume (8 ml) and incubate on ice 15 min
  10. Aliquot into pre-cooled tubes, 20 tubes with 200 l and 10 tubes with 400 l
  11. Quick freeze in liquid N2, using a different colored sharpie than the previous batch mark (see detail table this section) 200 l tubes with one slash and 400 l tubes with two slashes
  12. Store at -70°C
Using frozen competent cells
  1. Thaw in air at room temperature until just liquid, use 200 l per transformation
  2. Add DNA up to 20 l and swirl to mix
  3. Incubate on ice 30 minutes
  4. Heat shock at 42°C for 90 sec, then place on ice briefly
  5. Add 800 l LB and incubate at 37°C for 45 minutes
  6. Plate out under appropriate conditions
Notes:
  • Once iced, keep cells cool and pre-cool all tubes
  • Be gentle, don't vortex or roughly pipet cells
  • Use forceps to lower tubes into liquid N2 in case tubes leak