What is Electroporation?

The process of introducing nucleic acids (DNA or RNA) into eukaryotic cells by nonviral methods is defined as transfection.

Using various chemical, phospho-lipid or physical methods, this gene transfer technology is a powerful tool to study gene function and protein expression.

Transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., neagtively charged phosphate backbones of DNA and RNA) into cells with a negatively charged membrane.

Various chemicals such calcium phosphate and DEAE-dextran or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule and thus enable sucessful delivery inside the cells.

This makes it easier for the DNA or RNA:transfection reagent complex to cross the membrane, especially for phospho-lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer.

Physical methods like microinjection or electroporation simply punch through the membrane and introduce DNA directly into the cytoplasm. .

Electroporation Principle

In electroporation a high-intensity electrical field transiently permeabilizes the cell membrane, enabling uptake of exogeneous molecules from the surroundings.

This technique has been used to introduce nucleotides, DNA, RNA, proteins, carbohydrates, dyes and virus particles into prokaryotic and eukaryotic cells.

It provides a valuable and effective alternative to other physical and chemical methods for transfection.

Adopted from from BioRender

Requirements for Electroporation

Sterile:

1. Electroporation cuvettes

2. 6 well plates

3. Microtips

4. Growth medium

5. Cells (A 549 Cell line)

6. Trypsin EDTA

7. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).

8. DNA

Non sterile:

9. 70% IPA

10. Cotton balls

11. Electroporator

Cell Preparation

1. One – two days prior to electroporation, transfer the cells into 25cm2 flasks with fresh growth medium so that they will be 50–70% confluent on the day of the experiment.

For most cell lines the cell density will be 2–10 x 10^6 cells/ flask; about 1–20 x 105 cells are needed per electroporation.

2. Trypsinize the cells as described in previous experiment and scrap the cells if required with scrapper and transfer the cells along with the medium in to 15 ml centrifuge tube, and centrifuge the cells at 1300 rpm for 7 min at 40 C.

3. Discard the supernatant and re-suspend the cells in the required quantity of PBS (Phosphate buffer saline)

4. Count the cells using Neubar’s chamber and calculate the volume of the cell suspension to be used in the experiment (2 x 10^6 in 200 μl)

Electroporation Procedure

1. Set the parameter required for electroporation likes voltage, time pulse, no. of pulse (V 75 to 200, Pulse length 15 to 30 mili seconds)

Note: This is variable from instrument to instrument and cell type

2. Add plasmid DNA to the cuvettes (5 to 15 μg)

3. Then add 200 μl of cell suspension (containing 2 x 10^6 cells) to the 0.2 cm cuvette and tap the side of the cuvette to mix.

4. Place the cuvette in the shockPod. Push down the lid to close. Pulse once

5. Immediately after the pulse, transfer the cells to a 6 well plate and add 1.8 ml fresh media

6. Rock the plates gently to assure even distribution of the cells over the surface of the plate. Incubate the plates at 37 oC in 5% CO2 in a humidified incubator.

7. Add suitable concentration of antibiotics after 24- 48 hrs.

8. Do not keep the cells out for longer time after electric shock.

9. Do not rock the plates forcefully.