Ultra-Competent Cells Preparation

Prepare a buffer with the following composition

A	B	C
KCl	9.33 g	250 mM (18.65 g/L)
CaCl2 ⋅ 2 H2O	1.1 g	15 mM (2.2 g/L)
MnCl2 ⋅ 4 H2O	5.44 g	55 mM (10.88 g/L)
PIPES	10 mL from 0.5 M pH 6.7 Stock	10 mM (3.02 g/L)

Inoue Transformation Buffer Composition

Prepare Stock of PIPES 0.5 M Measure 7.56g and dissolve it in 40mL using a small beaker. Set a magnetic stirrer in the beaker and start stirring. Then, adjust the pH using a pH meter and 10M KOH solution (Or KOH pellets added one by one to the beaker). Final point is reached at 6.7 .

Prepare the transformation buffer solution

Place 400mL in a big beaker, drop a magnetic stirrer and add in the following order 9.33g , 1.1g and 5.44g .

Important! Wait until the previous salt has completely dissolved, the add the following. CaCl2 solubilization is highly exothermic, add carefully the pellets. MnCl2 should be added last.

After dissolving the salts, add 10mL , stir the solution and measure the final pH. It should be close to 6.7 . pH can not be adjusted after MnCl2 addition to avoid precipitation of the metal ions, thus it is extremely important that the PIPES pH is correct.

Transfer the solution to a big measuring cylinder and fill with deionized water (Clean freshly treated MiliQ if possible) up to 500 mL final volume.

Filter Sterilize the transformation buffer using a 0.45 μm filter. It is recommended to aliquot the buffer in 100 mL batches.

Storage. For long term storage freeze the transformation buffer at -20°C

Direct Utilization. Keep the filter-sterilized buffer at 4°C

Filter sterilize 1-5mL. It is recommended to use DMSO of the higher possible purity to ensure optimal competency in the cells.

Grow the required E. Coli strain in an LB Agar Plate , streaking the cells to obtain single colonies and incubate at 37°C

The next day, pick a single colony from the plate and inoculate 25mL . Incubate the cells at 180-240rpm . Recommended doing it early in the morning!

After the incubation, prepare 2 big E-Flasks with 200mL (SOB Media could be used instead). Inoculate one flask with 1mL of the starter E. Coli culture, the second flask receives 10mL of seed culture instead. Incubate the cells at 180-240rpm .

Cool down the centrifugue at 0-4°C to ensure it is already cold before starting.

Prepare an ice-bath in a styrofoam box and chill 100 mL of the transformation buffer on it for at least 30 minutes before starting the protocol.

Measure the Optical density at λ = 600 nm (OD600). ₆₀₀). When one of the culture reaches 0.55 OD₆₀₀. Stop the incubation and discard the other culture.

Take 200mL of the E. Coli culture at OD₆₀₀0.55 and split it into 4, 50 mL Falcon tubes.

Spin down the cells at: 2500x g,0-4°C

After centrifugation, place immediately the tubes on ice and always keep them there while working.

Discard the supernatant and remove the excess of media by tipping the tubes over paper towels.

Work under a flame or sterile hood when opening the tubes.

Add 16mL to each falcon tube and gently resuspend the cell pellet by swirling the tube. (Avoid pipetting or vortexing to keep cells integrity).

Spin down the cells at: 2500x g,0-4°C

After centrifugation, place the tubes on ice and discard the supernatant.

Add 4mL to each falcon tube and gently resuspend the cell pellet.

Then add 300µL to each falcon tube, and mix gently by inverting the tubes 3-4 times.

Incubate the tubes 0On ice for 0h 10m 0s .

Working as quick as possible, take one of the tubes and dispense 50-200µL aliquots of the suspensions into chilled, sterile 1.5 eppendorf microfugue tubes.

Immediately after dispensing the aliquots, close the tubes and freeze them on liquid nitrogen.