Competent cells Preparation

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