Transfection and expression of altered genes into cell lines are indispensable parts of genetic studies. There are many different methods for introducing genetic material into eukaryotic cells, usually by making pores in the cell membrane so that the foreign particles can enter into the cell. However, this may injure the cells and in turn lead to cell death. Other methods like gene gun or viral transduction may require specific equipment or facilities. Therefore, when choosing the suitable technique, one should take into account the transfection efficiency, resultant cell viability and the cost.
I have found that the GeneJuice® Transfection Reagent from Novagen can serve me well. Just seed the plates and wait until 80% confluence. Then prepare the GeneJuice®/serum-free medium/DNA mix as instructed. No need to remove the complete growth medium from the plates. Distribute the transfection mix drop-wise and evenly onto the plate. Incubate at 37°C, 5% CO2 for 24 hours. That’s it.
My labmates have compared transfection in the presence and absence of serum. Similar transfection efficiencies resulted. I have checked the cytotoxicity of the reagent by trypan blue staining. It caused ~12% cell death after 24-hour incubation in my cell line (COS7). That’s OK for me, as I removed the transfection mix afterwards and the cells grew well again before I harvested them for further investigation.
For some unknown reasons, transfection efficiency was apparently lower with deletion-containing plasmids as compared to the intact plasmid and a plasmid with a point mutation. I increased the amount of deleted plasmid DNA in the transfection mix, but it didn’t help much.
Another problem is that much of the transfection mix would adhere to the tube wall. According to the manufacturer’s instruction, 3 ul of GeneJuice® should be mixed with 100 ul of serum-free medium and 1ug of DNA for each well in a 6-well plate. Collectively, 6 wells should include 18 ul of GeneJuice, 600 ul of serum-free medium and 6 ug of DNA; each well is transfected with ~103 ul of the transfection mix. But I can never get sufficient volume unless I add an additional 20 ul of serum free medium to the mix to compensate the loss on the 1.5 ml tube wall.
However, apart from these drawbacks, the reagent works quite well and provides a stable performance. The simple protocol allows me to minimize the number of manipulation steps before I treat the cells. Moreover, the cytotoxicity is within an acceptable level. Therefore, I will keep using this reagent.
GeneJuice® Transfection Reagent From Novagen
Stable performance, cytotoxicity not very high, simple protocol, do not need to remove serum-containing medium before transfection.
Much of the transfection mix adheres to the tube wall. Need to scale up the volumes a little to compensate the loss.
The Bottom Line
Convenient to use; stable performance.