Table 1.
The experimental procedure to make an Fv library for affinity maturation by mammalian cell surface display
| Step | Day | Procedure |
|---|---|---|
| 1 | 1 | Prepare library plasmid DNA (≈200 μg) |
| 2 | 1 | Grow one 100-mm Petri dish of HEK 293 T cell to 60% confluence |
| 3 | 2 | The next day, transfect each dish with 5 μg of library DNA with Lipofectamine |
| 4 | 4 | 48 h after transfection, harvest cells (≈107 cells per dish) and perform flow cytometric sorting of 107 cells (10,000–20,000 cells per sec) |
| 5 | 4 | Collect 0.1% of cells (≈104) and recover plasmid DNA from cells |
| 6 | 4 | Transform the recovered plasmid DNA into E. coli |
| 7 | 5 | Pick E. coli colonies and grow overnight |
| 8 | 6 | Isolate plasmids and run sequencing reaction |
| 9 | 7 | Obtain sequence and plan next selection if necessary |