Protocols

Preparing electrocompetent bacteria

NOTE: The preparation of electrocompetent bacteria in room temperature (24°C) was investigated also to evaluate the increase of transformation efficiency [1].

1. Streak DH5α strain onto an LB agar plate and incubate at 37°C overnight.

2. Inoculate 5 mL of LB medium with a single colony of freshly grown E. coli and incubate at 37°C with vigorous shaking until the OD is approximately 0,25(+-0,05)

3. Collect bacteria in 5 microcentrifuge tubes (1mL for each tube).

NOTE: after incubation, tubes should be held in ice.

4. Pellet the cells by centrifugation at 9000g for 2 min at 4°C and discard the supernatant, leaving ~50 µL of it.

5. Resuspend the cells in 1 mL of ice-cold sterile distilled water (at 4°C)

6. Repeat steps 4 and 5. The cells need to be washed with water for three times. After the third washing all water needs to be discarded leaving the bacteria adhered on tube walls.

NOTE: since the centrifugation speed is low, bacteria adhere poorly and it is important to avoid discarding it with supernatant.

7. Resuspend the cells in 70 µL of ice-cold distilled water.

Electroporation

1. Add plasmids to suspension

NOTE: suspension should not be diluted with plasmids in more than 80ul.

CRITICAL: plasmids must be washed properly to reduce the concentrations of salts as much as possible, because salts might cause arcing in electroporator which leads to unsuccessful electroporation.

2. Prepare cuvettes for electroporation by leaving it on ice.

3. Insert suspension with plasmids into prechilled cuvette.

4. Take the cuvette and wipe its walls to avoid any condensate and immediately place the cuvette in electroporator.

5. Set the electroporation conditions on a Bio-Rad Gene Pulser to 2,5kV (Ec2 mode). Press the pulse button until a beep sounds and a time constant appears in the apparatus window.

NOTE: Voltage depends on the gap width of cuvette electrodes. In our experiments we used 0.2 mm cuvettes. 0.1 mm can be used also, but 1.8 kV voltage needs to be set.

6. After the cells have been pulsed, immediately add 1 mL of room temperature SOC medium and gently resuspend the cells in cuvette.

7. Transfer bacteria from cuvette to microcentrifuge tube and incubate at 37°C with vigorous shaking.

NOTE: Incubation time might vary depending on antibiotic and plasmids number.

NOTE: It might be difficult to collect all suspension from cuvette electrodes gap, you can do it by gently rotating cuvette and pipetting at the same time.

NOTE: Suspension could be transferred in 15 mL tube to increase efficient aeration and improve bacteria viability.

8. After the incubation, centrifuge the suspension at 9000g for 2 min at room temperature and discard supernatant leaving 50-80ul.

9. Resuspend bacteria and spread aliquots of the cells onto LB agar plates using glass beads.

10. Calculate grown colonies on LB agar plate. Make serial dilutions if needed to decrease colony-number.

Useful tips to increase the viability of cells and avoid arcing:

• Arcing may occur due to high concentration of salts or air bubbles.

• It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.

• Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%.

• Keep your cuvettes cold (we held them in a freezer (-23°C), however, lower volume of cells can freeze, so it would be beneficial to prewarm the cuvette for a few minutes before pouring the bacteria in).

• Do not touch the aluminum electrodes

• Even if your electroporation arced, it is possible that you might still have a transformed clone.

[1] Q. Tu, J. Yin, J. Fu, J. Herrmann, Y. Li, Y. Yin, A. F. Stewart, R. Müller and Y. Zhang, 2016, 6, 24648.