Test Accordian

Cuvette Electroporation Method

The NEPA21 is most commonly used with electroporation cuvettes. We have an in-house written protocol available to any lab that owns a NEPA21 system. Please ask us for this document.

The NEPA21 cuvette protocols usually use 3- or 4-phases of electrical pulsing, including a Poring Pulse Phase to “open holes” in the membrane, followed by a Transfer Pulse Phase to move the charged molecules (usually DNA, RNA or proteins) into the cell. Both phases are essential for high cell viability and transfection efficiency.

Advantages:

• No requirement for special buffers or expensive reagents
• Fully adjustable electrical pulsing parameters
• Non-capacitor design for handling physiological salt conditions
• Can be used to transfect large plasmids (10+ kb)
• Works with proteins, RNA, and gene editing complexes
• Proven on hundreds of cell lines and cell types.

Key publications: The NEPA21 has been referenced in more than 800 publications since 2012. Most of these use 2 mm electroporation cuvettes. We would be happy to help you find the most pertinent publications for your lab’s particular experimental focus.

Using the NEPA21 cuvette method:

Ask us for both a written protocol and for our suggested pulsing parameters for your lab’s cells of interest. These can be supplied to any NEPA21 lab.

Cell Types:

Hundreds of cell lines and cell types have been demonstrated to work with the NEPA21 system. Below is a quick summary: click here for an up-to-date list of dozens of cell types with images and expected results.

• Primary cells (including stem cells, neurons, and many more)
• Cell lines (all common and many obscure)
• Immune cells (including B and T lymphocytes)
• Cell clusters (including organoids and dissociated cells from tissues)
• Walled cells (including Chlamydomonas and Diatoms)
• Marine protists (e.g., Euglena)

Electrode Types:

CU500 Electroporation Cuvette Chamber

• Accepts industry-standard electroporation cuvettes
• Ask about samples of 2 mm gap electroporation cuvettes here.

2 mm gap Electroporation Cuvette

• 100 μL volume
• buffer type: Opti-MEM
• most commonly recommended
• 1 x 10⁶ cells
• 10 μg of plasmid DNA
• target Impedance reading is 30 Ω to 55 Ω

Tips for Success:

1. Please ask us for a spreadsheet with recommended, field-tested, parameter sets to try. When first optimizing a new cell line or cell type, use ALL of the recommended pulsing parameters for that cell type.
2. Always record output electrical data – especially the IMPEDANCE value. We are happy to offer suggestions for further optimization, but we can only help you interpret your results when the electrical data is available.
3. We recommend performing the transfection in serum-free (or at minimum serum-reduced) Opti-MEM. The cells should be washed at least twice to ensure good electric-field formation.
4. Use a high quality source for reagents. In the case of DNA plasmids purify using an endotoxin-free kit. For gene editing reagents we recommend using the Cas9 protein instead of mRNA.
5. If you are using your own plasmid vector, please ensure that it has already been fully qualified and works for other transfection methods. Otherwise, ask us about our control DNA, which contains a general GFP expressing vector for mammalian cells (pCMV-EGFP or pCAGGS-EGFP). In this case, please use it for each of the parameters. The amount that should be used is in the protocol sheet, typically 10 μg in 100 μL of cells at 1 x 10⁶ concentration.
6. Avoid bubbles when loading into the cuvette.
7. Do not lower the DNA amount until pulsing parameters have been fully optimized.
8. It is best to dry down the DNA pellet and resuspend in ddH2O to avoid any molecules that may affect the ionic conditions (such as EDTA).
9. Immediately plate cells after electroporation and before pulsing the next cuvette. Don’t allow cells to remain in the cuvettes any longer than necessary following electrical pulsing.
10. **Imperative** It’s important to properly resuspend cells within the cuvette to eliminate clumping that can occur both before and after electric pulsing. This clumping can drastically reduce cell viability and/or transfection efficiency, and often leads inconsistent data. Please use a gel-loading pipette tip to load and retrieve all cells from the cuvette. Repeated, gentle pipetting through the small-bore tip will help break up clumps.