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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°C. （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°C 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°C.

2. 1 ml culture was inoculated in 100 ml (1:100dilution) SOB. Incubate at 37°C 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°C.

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 4ºC. Drain the pelleted cells thoroughly by inverting the tubes on paper towels, and rapping to remove any liquid. A micropipette can be used to draw off recalcitrant drops.

5. Resuspend each of the cell pellet in TB by pipetting up and down very gently in 1 ml of TB. After the cell pellet was pipetted to single cell, another 9ml of TB was added. Combine the cells in the 2 tubes. Incubate the cells on inc for 15 min.

6. Collect the cells by centrifugation at 750-1000 g for 10 min at 4°C.

7. Resuspend each of the cell pellet in TB by pipetting up and down very gently in 1 ml of TB. After the cell pellet was pipetted to single cell, another 7ml of TB was added. (to 1/12.5 of the original volume)

8. Add 280 µl of DMSO per 8 ml resuspended cells. Mix gently by swirling, and store the suspension on ice for 15 min.

9. Add another 280 µl of DMSO per 8 ml resuspended cells. Mix gently by swirling, and then return the suspensions to an ice bath.

10. Distribute aliquots 100 µl with chilled, sterile pipette tip into chilled 1.5 ml microcentrifuge tubes.

11. Flash freeze in liquid nitrogen, then place at -70°C.