Monitoring of Spontaneous Survival Plasmid Loss by Hirt Supernatant Mediated Bacterial Transformation
| Days without selection | Bacterial transformant per 106Clone 12 cells | Plasmid loss (k) per day (%) |
| 0 | 628 | |
| 21 | 38 | 13.4 |
| 28 | 23 | 13.2 |
| 35 | 16 | 9.7 |
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Clone 12 cells were plated at the beginning of the experiment in DMEM without hygromycin B. Cells were continuously passaged throughout the entire experiment. Low-molecular weight DNA present in Hirt supernatants was collected (Hirt 1967) at the indicated time points. All Hirt supernatants were normalized by addition of 1 ng of a chloramphenicol-resistant plasmid prior to the beginning of cell extraction. Each Hirt supernatant transformation of bacteria was plated both on chloramphenicol and ampicillin plates. Values in the table were normalized to the number of colonies counted on the chloramphenicol plates.
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↵Calculated according to 2.303 log (N0/N) = kT, where N0 = % AmpR colonies at time zero (100%), N = % AmpR colonies remaining after propagation for T days without hygromycin B selection and k = % loss of plasmid per day.
