Electroporation—Troubleshooting

Here are possible causes for low transfection efficiency using the Neon device:

1. Sub-optimal electrical parameters
2. Plasmid preparation containing high salt
3. Plasmid larger than 10 kb
4. Plasmid concentration too low
5. Cells are stressed, damaged, or contaminated by Mycoplasma
6. Cell density too low or too high
7. Cells with high passage number

Here are possible causes for low transfection efficiency using the Neon device:

1. Sub-optimal electrical parameters
2. Poor plasmid quality such as endotoxin contamination
3. Plasmid preparation containing high salt
4. Plasmid quantity too high
5. Cells are stressed or damaged
6. Multiple uses of the same Neon tip
7. Microbubbles in tip, causing arcing

Yes. We recommend verifying the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid. The content of “nicked” DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in a significant decrease in transfection efficiency.

Yes. To check the quality of your DNA, we strongly recommend determining the A260:A280 ratio. It should be at least 1.6 for a good DNA preparation.

No, the precipitation is irreversible. Please contact Technical Support to obtain a replacement, if the Neon kit was purchased within 1 year. 

For large plasmids, it is important to prepare highly concentrated plasmid. For the control plasmid, 0.5 mg is used for 10 mL electroporation and the size of the control plasmid is ~5.5 kb. Let’s say the large plasmid being used is 50 kb. This is almost 10 times larger than the control, so you would have to use 10 times more plasmid to compensate for the molecular number. So you would use 5 mg in 10 mL electroporation. To cover this large amount of plasmid, the plasmid concentration should be over 5 mg/mL. One thing to keep in mind is that when you add large amounts of plasmid, it can damage cells due to toxicity of the plasmid sample itself. Thus, we recommend optimizing, starting with a plasmid amount that is less than the amount you came up with, and then moving up in plasmid amount while checking both viability and transfection efficiency. 

There are several possible reasons for this. Monocytes and macrophages respond to very low levels of endotoxin (LPS), which could have been introduced with your plasmid DNA. Make sure that you use plasmid DNA that has been purified by anion-exchange chromatography, such as our PureLink HiPure Plasmid Purification Kits. If you still observe activation, you may subject your plasmid to a second round of anion-exchange chromatography purification. If you still get activation, the plasmid itself may contain sequences that stimulate the production of Interferon gamma. It is also possible that certain components in your culture medium, including the FBS batch you are using, may cause activation. Please make sure that none of these components activates your cells. The procedure for isolating your monocytes is also important. We recommend negative rather than positive selection, as it leaves the monocytes “untouched” by antibodies. Our electroporation buffers are guaranteed to be endotoxin-free and do not cause monocyte/macrophage activation in our hands.

To determine the Neon transfection efficiency for siRNA, we recommend transfecting the cells with a fluorescent-labeled negative control siRNA (BLOCK-iT™ Fluorescent Oligo, Cat. No. 13750062) and measuring the transfection efficiency by the percentage of fluorescent stained cells among viable cells. However, keep in mind that there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescence detection with a microscope does not distinguish the siRNA that enters the cell from the siRNA that sticks to the cell membrane. To measure transfection efficiency more accurately, one needs to transfect the cells with a positive control siRNA, such as one that targets a housekeeping gene, and measure the knockdown of target RNA or protein.

Arcing could be caused by high salt in the DNA preparation, high cell density, and bubble formation at the end of the tip piston. Please look at the bottom of the piston for microbubbles that may have formed due to hasty pipetting technique or samples that were mixed too vigorously. You should pipette the sample in a slow, smooth, and continuous motion to avoid air uptake.

If you are troubleshooting all of the above and still experiencing arcing issues, it is possible that you are using an older lot of tips where the resistance of the piston in the tip may be low, causing bubble formation and arcing. If this is the case, we recommend switching to a new batch of tips.