Can nicked DNA lead to reduced transfection efficiency in Nucleofection® experiments?


Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded.

Compare undigested plasmid to plasmid DNA digested with a suitable single cut restriction enzyme to linearize. Supercoiled plasmid will run faster than linear. Nicked plasmid will run slower than linear. The content of nicked DNA (on an agarose gel visible as retarded band in the undigested sample) in your DNA preparation should be below 20%. Higher contents of nicked DNA result in significant decrease of transfection efficiency. Up to three fold lower transfection efficiencies have been observed. You can find further information on this topic and on troubleshooting in our webinar: "Tips and Tricks for Successful Transfection Experiments"

Research Areas:
Gene Expression