Endotoxin free plasmid maxiprep kit

To generate stable cell lines expressing wild-type (WT) or mutant G9a, the pHRCMVpuro-Sin8 vector containing a puromycin resistance cassette was introduced simultaneously with a threefold excess molar ratio of the G9a expression vector into the G9a-null ES cells by either standard electroporation or by nucleofection. Nucleofection (1 μg of plasmid DNA per 10⁶ cells) was performed using the Amaxa 4-D Nucleofector System (Lonza) with the P3 Primary Cell 4-D Nucleofector X Solution and the CG-104 program. Plasmid DNA used for nucleofection was purified using the Endotoxin-free Plasmid Maxiprep Kit (Qiagen) according to the manufacturer’s instructions. The stable transfectant clones expressing mutant or WT G9a were selected in ESC medium containing puromycin (1 μg/ml). All mutations were confirmed in the selected cell clones by sequencing.