Table 2.

Troubleshooting guide.

Step	Problem	Cases	Suggestions
2.3	Low cell yield	Cells under-confluent Culture plates not rinsed thoroughly Poor cell health	Allow cells to expand longer Rinse culture plates thoroughly to maximize cell yield Increase the number of round culture dishes Thaw a new batch of cells Check for mycoplasma contamination Do not overgrow cells (keep culture in logarithmic growth phase)
3.5	HeBS/plasmid complex solution is not cloudy	Plasmid solution added too quickly Incorrect pH of 2× HeBS Not vortexed vigorously	Plasmid solution needs to be added slowly and dropwise to 2× HeBS Ensure HeBS has a pH of 7.08 (± 0.01) Vortex as vigorously as possible
3.5	HeBS/plasmid complex solution has precipitated out	Incorrect calcium chloride used	Make sure to use Cacl2·2H2O (MW-147.01 g/mol)
5.7	Clogged filer	Too much debris in supernatant	Replace filter Centrifuge supernatant twice Use a filter with a larger binding surface area Use a series of filters with decreasing g pore size
5.14	Low tire	Poor plasmid quality Unhealthy cells Mustang Q XT5 column not equilibrated	Check quality of plasmids Ensure only healthy cells are used for production Re-equilibrate the Mustang Q XT5 column following part 6: Mustang Q XT5 column equilibration and storage Check the ultracentrifugation tubes are a suitable plastic (e.g., polyallomer)