TITLE: Ultra-Competent Cells Preparation 
AUTHORS: jorge.fernandez

[DESCRIPTION]
Adaptation of the Inoue protocol for competent cells preparation. 

Expected transformation efficiency of 108 colonies per μg of plasmidic DNA.

Original Source: doi:10.1101/pdb.prot101196 

[STEPS]
SECTION: Preparation of Transformation Buffer &amp; Reagents
1. Prepare a buffer with the following composition
 

SECTION: Preparation of Transformation Buffer &amp; Reagents
1.1. Prepare Stock of PIPES 0.5 M

Measure  7.56 g  and dissolve it in  40 mL 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 pH 6.7 . 
 


SECTION: Preparation of Transformation Buffer &amp; Reagents
1.2.  Prepare the transformation buffer solution

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

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.


SECTION: Preparation of Transformation Buffer &amp; Reagents
1.3. After dissolving the salts, add 10 mL , stir the solution and measure the final pH. It should be close to pH 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.
SECTION: Preparation of Transformation Buffer &amp; Reagents
1.4. 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. 
SECTION: Preparation of Transformation Buffer &amp; Reagents
1.5. 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 

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

SECTION: E. Coli Cultivation
3.1. The next day, pick a single colony from the plate and inoculate 25 mL . Incubate the cells  at     . Recommended doing it early in the morning!

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


 


SECTION: Previous Preparation of Competent Cells
5. Measure the Optical density at λ = 600 nm (OD600). When one of the culture reaches 0.55 OD600. Stop the incubation and discard the other culture.
SECTION: Preparation of Transformation Buffer &amp; Reagents
2. Filter sterilize 1-5 mL. It is recommended to use DMSO of the higher possible purity to ensure optimal competency in the cells. 

SECTION: Previous Preparation of Competent Cells
4. Cool down the centrifugue at 0-4 °C  to ensure it is already cold before starting. 

SECTION: Previous Preparation of Competent Cells
6. Take 200 mL of the E. Coli culture at  OD600 0.55 and split it into 4,  50 mL Falcon tubes.
Spin down the cells at:2500 x g, 10 min, 0-4 °C 

SECTION: Previous Preparation of Competent Cells
4.1. 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.
SECTION: Previous Preparation of Competent Cells
7. 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 16 mL to each falcon tube and gently resuspend the cell pellet by swirling the tube. (Avoid pipetting or vortexing to keep cells integrity).
SECTION: Previous Preparation of Competent Cells
8. Spin down the cells at:2500 x g, 10 min, 0-4 °C

After centrifugation, place the tubes on ice and discard the supernatant.
SECTION: Previous Preparation of Competent Cells
9. Add 4 mL to each falcon tube and gently resuspend the cell pellet. 

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

Incubate the tubes on ice for 10 min .

SECTION: Previous Preparation of Competent Cells
10. Working as quick as possible, take one of the tubes and dispense 50-200 µL 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.