Jan 31, 2023
Ultra-Competent Cells Preparation
- 1Uppsala University, Mikrobial Kemi
External link: http://doi:10.1101/pdb.prot101196
Protocol Citation: jorge.fernandez 2023. Ultra-Competent Cells Preparation . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7yd48gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Standard Laboratory Protocol.
Created: January 31, 2023
Last Modified: January 31, 2023
Protocol Integer ID: 76131
Keywords: competent cells preparation, plasmidic dna, cell, dna, adaptation of the inoue protocol, inoue protocol, preparation, adaptation
Abstract
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
Materials
- Big 1L E-Flasks, x2
- 50 mL Centrifugue Falcon Tubes
- Centrifugue with 50 mL Tubes adapter
- Incubator
- MnCl2 • 4H2O
- CaCl2 • 2H2O
- PIPES Buffer
- DMSO
- KOH
- LB Media (550 mL)
Preparation of Transformation Buffer & Reagents
Prepare a buffer with the following composition
| Component | Ammount for 0.5 L | Final Concentration | |
| 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 7.56 g of PIPES and dissolve it in 40 mL of deionized H2O 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 .
Note
STORAGE of PIPES Stock. The prepared PIPES buffer can be stored for further use. In this case, filter sterilize the solution with a disposable 0.45 μm filter, and freeze it at -20 °C.
Prepare the transformation buffer solution
Place 400 mL of deionized H2O in a big beaker, drop a magnetic stirrer and add in the following order 9.33 g of KCl , 1.1 g of CaCl2 ⋅ 2 H2O and 5.44 g of MnCl2 ⋅ 4H2O .
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 10 mL of PIPES 0.5M pH 6.7 , 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 1-5 mL of pure DMSO . It is recommended to use DMSO of the higher possible purity to ensure optimal competency in the cells.
E. Coli Cultivation
6h
Grow the required E. Coli strain in an LB Agar Plate, streaking the cells to obtain single colonies and incubate at 37 °C Overnight
The next day, pick a single colony from the plate and inoculate 25 mL of LB . Incubate the cells at 180-240 rpm, 37°C, 06:00:00 . Recommended doing it early in the morning!
After the incubation, prepare 2 big E-Flasks with 200 mL Sterile LB (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 180-240 rpm, 18-22°C Overnight .
Note
E. Coli cells grown at low temperatures has been shown to improve it's transformation efficiency, likely due to changes in the membranes composition. However they grow slow and they can take up to 36 hours to grow at the required OD.
It is recommended to start the cultures in the evening of the previous day. Two flasks are used to ensure that at least one of them has the proper OD.
If there are no incubators with temperature control capable of achieving 18 °C (optimal), they can be grown at room temperature in the lab (Normally fluctuating between 20-24 °C ).
Previous Preparation of Competent Cells
30m
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). When one of the culture reaches 0.55 OD600. Stop the incubation and discard the other culture.
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, 0-4°C, 00:10:00 , (Approx 3,900 rpm for standard lab centrifugue).
10m
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 of Transformaion buffer 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:2500 x g, 0-4°C, 00:10:00 , (Approx 3,900 rpm for standard lab centrifugue).
After centrifugation, place the tubes on ice and discard the supernatant.
10m
Add 4 mL of Transformaion buffer to each falcon tube and gently resuspend the cell pellet.
Then add 300 µL of Sterile DMSO to each falcon tube, and mix gently by inverting the tubes 3-4 times.
Incubate the tubes On ice (0 °C) for 00:10:00 .
10m
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.
Note
Freezing on liquid N2 could be avoided by it enhances the competency of the cells specially during long term storage. Alternatively the aliquots can be kept on ice for some minutes and quickly moved to a -70 °C freezer (or lower temperatures).