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Preparing Competent E. coli cells

Protocol from the Benfey Lab

Yields ~200 50uL tubes of cells

DAY 1:

1. Streak antibiotic-free LB plate with stock cells (protocol works with DH5-¿, TOP10, DB3.1, and JM109 strains). Incubate plate at 37 ¿C overnight.

DAY 2:

1. Prepare 250 ml 2X YT broth in 500 ml flask:

4 g Bacto tryptone
2.5 g Bacto yeast extract
1.25 g NaCl
Water to 250 ml
pH to 7.0 with NaOH

2. Autoclave 2X YT broth, 500 ml flask (for secondary culture), 50 ml graduated cylinder (for TS buffer), and 150 ml beaker (for TS buffer, with stir bar).

3. Start an overnight culture in 4mL YT broth. Shake at 37C

DAY 3:

1. Transfer 2 ml of primary culture into 500 ml flask with 200 ml room temperature 2X YT broth (reserve remaining 2X YT broth to use as blank for OD600 reading). Shake at 37C until OD600 = 0.3-0.6 (2-3 hours).

2. While incubating secondary culture, prepare 15 ml TS buffer (TSB):

7.5 ml 2X YT broth (final concentration 1X)
129 µl 5M NaCl (final concentration 43 mM)
150 µl 1M MgCl2 (final concentration 10 mM)
150 µl 1M MgSO4 (final concentration 10 mM)
1.5 g polyethylene glycol (final concentration 10%)
750 µl DMSO (final concentration 5%)
3 ml glycerol (final concentration 20%)
Water to 15 ml
Filter sterilize with 0.2 or 0.22 µm filter and refrigerate until needed

3. Divide secondary culture into four 50 ml tubes. Centrifuge in Jouan 20 min at 4 ¿C. Discard supernatant. Keep cells on ice from here on.

4. Resuspend cells in 10 ml cold TSB (2.5 ml per tube). Pool cells into a single tube. Incubate on ice 10 min.

5. Aliquot into 1.5 ml tubes (50 µl each), on ice. Freeze immediately in liquid N2. Store at -80 ¿C.

6. Test for antibiotic resistance by plating tubes of cells (untransformed) on LB/amp and LB/kan plates.

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