Try to keep the DNA amount for Nucleofection™ quite low (e.g. 1 µg plasmid DNA per 100 µl reaction) since higher DNA amounts might cause increasing toxicity and mortality. It is also important to use highly purified DNA, e.g. by using Endofree™ Plasmid Kits (Qiagen). The A260:A280 ratio should be at least 1.8 for Nucleofection™.