Design Your Own Electroporation Protocol Episode 5 -
Considering Transfectant Amount
By Michelle M. Ng, Ph. D.
This is the fifth post in a series where we are providing tips for developing or improving your own electroporation method.
Transfectant is the molecule of interest that you wish to deliver to your target cells or tissues. The amount of transfectant used in your experiment can impact transfection efficiency. Increasing the amount of transfectant can boost efficiency up to a point; determining the optimal amount to use by running a titration experiment can be helpful. To do this, you would want to test a range of transfectant amounts while keeping cell number, buffer, and pulse generator settings the same. Then at the completion of this titration experiment, the transfection efficiency and cell viability for each transfectant amount may be assessed.
Note: The optimal transfectant amount may vary from one construct to another, so it is always a good idea to run a titration experiment each time you begin transfecting a new nucleic acid, protein, or other molecule of interest.
Typical ranges of transfectant for mammalian cells are as follows:
- DNA: 5 to 20 µg/ml (although sometimes increasing to high concentrations such as 50 to 100 µg/ml may enhance transfection efficiency)
- siRNA or miRNA: 5 to 100 nM final concentration. The optimal concentration will be a balance of achieving knockdown vs. minimizing off-target effects. Depending on the potency of the particular siRNA or miRNA and the types of stabilizing modifications on the molecule, more or less may be needed. Modified siRNA typically are used at a final concentration in the 5 to 20 nM range whereas unmodified are more commonly used at a final concentration in the 50 to 100 nM range.
- mRNA 1 to 10 µg per million cells
- Protein 1 to 10 µg per million cells
Tip: Dilute your transfectant in sterile nuclease free water to prevent arcing due to salts or reduced efficiency caused by EDTA or Tris containing buffers.
Coming next is Episode 6 - Optimizing Cell Density
Click here to visit our Protocol Database, for electroporation protocols searchable by, system, cell/tissue type, application/transfectant, and citation.