Top10 competent cells

Preparation of competent TOP10 cells using rubidium chloride (RbCl₂)

(modified from Naldini’s protocol)

· Inoculate a single colony of TOP10 from an LB plate in 10mL of LB medium or 2x YT (no antibiotics). Incubate ON at 37°C with vigorous shaking (approximately 225 rpm)

· On the following day, use the entire overnight culture to inoculate 500 mL of LB medium or 2x YT (no antibiotics). Grow cells in a 1L baffled flask until the A₆₀₀ reaches 0.4 - 0.6 (A₆₀₀ 0.5 in typically 2-3 hours). A 1L flask is necessary for proper aeration during growth.

NOTE: always treat competent cells as gently as possible, as they are very sensitive to handling and elevated temperature.

NOTE: leave cells at 37°C (never take them out!) and pause shaking for OD measurements.

SOLUTIONS

2x YT for 1L

Bacto Tryptone 16g

Bacto Yeast extract 10g

NaCl 5g

Dissolve in 800 mL oh H₂O pure. Bring solution to the volume, filter and sterilize by autoclaving.

TFBI for 250 mL

30mM potassium acetate 0.74g

10mM CaCl₂ 1.25 mL of 2M

50mM MnCl₂ 2.47g

100mM RbCl₂ 3.023g

15% Glycerol 37.5 mL

Adjust pH 5.8 with 1M acetic acid (glacial acetic acid). Filter-sterilize 0.22 um.

NOTE: take care when titrating this solution; if you overshoot and try to bring the pH back up with hydroxide, the manganese will precipitate.

TFBII for 100mL

10mM MOPS or PIPES (pH 7.0) 0.335g

75mM CaCl₂ 3.75 mL of 2M

10mM RbCl₂ 0.12g

15% Glycerol 15 mL

Adjust pH to 6.5 with 1M KOH. Filter-sterilize 0.22 um.

IMPORTANT: Store TFBI and TFBII solutions at 4°C. They must be ice-cold for the procedure.

From now, all steps are performed in the cold room at 4°C. Always carry with you an ice bucket and keep cells on ice. All pipets, tips, tubes and flasks must be pre-chilled. Cells must be treated gently: use a 25 mL stripette for all resuspension steps (NEVER use vortex).

· Cool down cells on ice for 10 min

· Pellet cells at 3,000 rpm g for 10 minutes at 4°C.

· Discard medium and gently resuspend the pellet in 150 mL of TFBI (pre-chilled) and pool all resuspended cells in one bottle. Leave on ice for 1 hour.

· Centrifuge cells at 3,000 rpm for 10 minutes at 4°C and discard supernatant.

· Gently resuspend pellet in 20 mL TFBII (pre-chilled).

· Aliquot 200 µL of cells into pre-chilled tubes. Quick-freeze the tubes in liquid N₂ and store at -80°C.

NOTE: cations such as Rb⁺², Ca⁺² and Mn⁺² neutralize the negative charge presents on the outer side of the bacterial wall. DNA molecules can now approach the bacterial wall.

Check transformation efficiency

Transform 100 μl of your new competent cells with 10 ng of your favorite plasmid (choose a gene codifying a protein easy to produce in E.coli. Complicated proteins affect the transformation efficiency, they are difficult to produce and fold so bacteria are not so happy), plate and incubate overnight at 37°C.

Count CFU and calculate efficiency using the following formula:

Efficiency = # of colonies per μg = # of colonies X 4 X 10⁴.

You should obtain 1-5 X 10⁷/μg from competent cells after one freeze-thaw cycle.