Simple and Efficient Method (SEM) to Make Competent Cells

Preparation of Frozen Competent of DH5α

1) 250 ml TB solution

10mM Pipes or 10 mM Hepes, 0.65g

15 mM CaCl₂, 0.55g

250 mM KCl, 4.66g

*55 mM MnCl₂, 2.47g

All the components except for MnCl₂ were mixed and the pH was adjusted to 6.7 with 0.1 N HCl (If you overshoot, do not readjust the pH by adding base, instead, discard the solution and begin again). Then 2.475 g MnCl₂ was added to the solution and dissolved. The solution was sterilized by filtration through a prerinsed 0.45 um filter unit. Dispense the solution into 50-ml aliquots and stored at 4℃.

2) Other Solution:

A. 100 ml of 250 mM KCl solution with ddH₂O, non-autoclaved: 1.86 g of KCl in 100 ml ddH₂O.

B. 10 ml of 1M MgCl₂ 1 M MgSO4 with ddH₂O, autoclave. (Prepare 1M MgCl2 first, then add MgSO₄ into it)

C. 50 ml of 1 M glucose with ddH₂O, filter, aliquot in 1.5 ml, store at -20℃

D. For 100 ml SOB medium: （Amount/volume）

Bacto tryptone: 2 g

Bacto yeast extract 0.5 g

NaCl 0.05 g

Shake until the solutes have dissolved.

Add 10 ml of a 250 mM solution of KCl

Adjust the pH to 7.0 with 5N NaOH (~0.2 ml)

Adjust the volume of the solution to 100 ml with ddH₂O

Sterilize by autoclaving for 20min at 15 lb/sq. in on liquid cycle

Just before use, combine the medium with 1 ml of 2 M Mg²⁺ (20 mM final)

E. SOC medium:

SOC medium is identical to SOB medium, except that it contains 20 mM Glucose.

3) A autoclaved 500 ml flask.

Method: Do it on ice!!!

1. Using a autoclaved yellow tip, streak DH5α directly from a frozen stock into 2 ml SOB, shake O/N at 37℃.

2. 1 ml culture was inoculated in 100 ml (1:100dilution) SOB. Incubate at 37℃ with moderate agitation for 2~2.5 hours, the cell density is 4~7×107 viable cells/ml or OD₆₀₀=0.5. To monitor the growth of the culture, determine the OD₆₀₀ every 20~30 min.

Or:

1. Using a sterile yellow tip, streak DH5α directly from a frozen stock onto the surface of an SOB agar plate. (Don’t thaw the frozen stock of bacteria, so the single tube of frozen stock can therefore be used many times.) Incubate the plate for 16 hours at 37℃.

2. Transfer four or five well-isolated colonies into 1 ml of SOB. The colonies should be 1-2 mm in diameter. Disperse the bacteria by vortexing at moderated speed, and then dilute the culture in 100 ml of SOB in a 500 ml flask.

3. Collect the culture into 2 of 50 ml Falcon tubes and chill on ice for 10-15 min.

4. Pellet the cells by centrifugation at 750-1000 g (2000-3000 rpm n a clinical centrifuge) for 12 min at 4℃.