Cell Preparation

E.coli host cells were cultured by inoculating 0.5 ml of overnight-grown fresh cells，or cells freshly thawed from a frozen cell bank，into 25 ml of R2Bl medium.The cells were allowed to grow in an incubator shaker（180 rpm）at 37℃overnight（18 to 24 h）to the stationary phase.The optical density（OD680）of the culture was measured at harvest（measurement at 1∶50 dilution）.The culture volume corresponding to a total 4 OD680，equal to approximately 1.5×109cells（e.g.，400 μl of cell culture at 10 OD680/ml），was transferred to a microcentrifuge tube and spun briefiy（30 s at 10，000 rpm，4℃）.The supernatant was discarded，and the resulting cell pellet was used for bacterial DNA isolation.

Figure 1　General description of bacterial PCN（plasmid copy number）assay procedure(https://www.daowen.com)

If needed，bacterial cell pellets or cell cultures can be kept at-20 to-80℃for at least 1 year prior to DNA isolation（frozen cultures should be thawed，and then spun as described above，before further processing）.Alternatively，a short-term storage of freshly harvested bacterial cell culture is possible for at least 5 days at 4℃.We have found that storing the cells as mentioned above will not affect the final plasmid copy number.