Experimental Outline for Targeting ES Cells - Step by Step
General Considerations
Plan out a flow chart from day 1 to day 16–19.
Note: Depending upon the growth of the ES cells, the days may have to be shifted. Electroporation, screening, picking and preparation of DNA will take 2–3 weeks, including weekends.
| Day No. | Application |
| 1. | Prepare culture plates for plating |
 | Prepare PMEF Feeder cell plates for ES cell expansion & electroporation, if applicable |
| 2. | Check PMEF Feeder cell plates, and thaw ES cells |
| 3. | Feed ES cells; passage if required |
| 4. | Feed ES cells; passage if required. Prepare Targeting Vector: Linearize ~100 µg of targeting vector (each electroporation requires 15–30 µg of linearized vector), and precipitate with EtOH (no need to phenolize). |
| 5. | Electroporation of ES cells |
| 6-10. | Select for ES cell transformants |
| 9-11. | Pick ES Cells |
| 12-13. | Feed picked ES cells |
| 14. | Freeze ES cell clones & retain duplicate wells to grow ES cells for DNA isolation Preparation of Genomic DNA for Southern blot analysis |
| 16-19. | Recover clones following analysis using RESGRO Culture Medium |
| |
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