Transformation into E. coli and Bacteria by Electroporation

Transformation into E. coli and Bacteria by Electroporation

APPLICATIONS

– Transformation data of bacteria (E. coli) –

We measured gene transfer efficiency using the ELEPO21 in Gram-negative bacteria. Competent cells were prepared as usual from E. coli DH5α. The competent cells were mixed with pUC19 DNA, and a 20μl aliquot (containing 10^9-10^11 cells and 10 pg DNA in 10% glycerol solution) was transferred to the 1 mm gap electrode cuvette (EC-001S, Nepa Gene). The cuvette was set in the chamber connected to the ELEPO21, and delivered 3-step pulses as described below. All steps were done on ice. After electroporation, the cells were plated on LB agarose medium containing ampicillin, and colonies formed were counted. Transformation efficiency was expressed as a number of colonies per ug plasmid DNA used.

 

[ELEPO21 pulsing conditions, Fig. 1]
Poring Pulse (voltage: 2,000 V, pulse length: 2.5 msec, pulse interval: 50 ms, number of pulses: 1, polarity: +)
Transfer Pulse (voltage: 150 V, pulse length: 50 msec, pulse interval: 50 ms, number of pulses: 5, polarity: +/-)
To evaluate the above results, we measured gene transfer efficiencies using a conventional electroporator (ECM630, BTX) that deliver a single exponential pulse as described below.

 

[ECM630 pulsing conditions, Fig. 2]
Single pulse (voltage: 2,000 V, resistance: 200 ohms, capacitance: 25 uF, number of pulse: 1)

 

 

Fig. 1: ELEPO21 pulse shape Fig. 2: ECM630 pulse shape

 

Fig. 3:

 

[Experimental results]:

The above cell suspensions (sample resistance value: 7.7 K ohms) were used for electroporation. The tranformation efficiency obtained by the ELEPO21 electroporator was approximately 5 times higher than that by the ECM630 electroporator (Fig. 3).

*The values are averages of repeated experiments.
*The optimum pulsing conditions were used for ELEPO21 and ECM630.
*cfu: colony forming unit.

PUBLICATIONS

Electroporation

Drug Delivery and Transfection

Electro Cell Fusion

Fluorescent Staining

Single-Cell/Micro-Particle Transfer

Cell Freezing

Mechanical Vibration