Digoxigenin nick translation kit

Fluorescence in situ hybridization was performed to determine the episomal sites and gene copy number of the pEMEα vector. Monoclonal cells transfected with pEMEα and showing different expression levels were collected. eGFP served as a probe and was labelled using a Digoxigenin‐Nick translation kit (Roche, Mannheim, Germany). Samples were counterstained with 1 μg/ml 4′,6′‐diamidino‐2‐phenylindole before examination under a Leica DMRB fluorescence microscope with a Leica DC 300 f camera. Approximately 50 visual fields were examined, and mean copy numbers were calculated. Fifty metaphase plates were analysed by FISH for each vector type.

To verify the status of vectors within the selected stable PEF cells, FISH was performed as described previously at 20 generation post-transfection [13]. eGFP probe was labeled using a digoxigenin-nick translation kit (Roche, Mannheim, Germany). The samples were counterstained with 1 μg/mL of 4',6'-diamidino-2-phenylindole and further analyzed using a Leica DMRB fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Approximately 10 fields were observed.