14. We usually freeze ES cells in cryovials at a density of 5-10 x 106 cells/mL of freezing medium. A single 10-cm dish usually gives approx three cryovials each containing 1 mL of cells.
15. To freeze cells from different-sized dishes, proportionally altered volumes of cell medium are used (for example, a 6-cm dish requires 1 mL, and a 3.5-cm dish requires 0.5 mL).
16. It is important to note that the cells should be frozen down gradually (in a styrofoam box or isopropanol container), and not to be kept at -70°C for too long a period of time. Cryovials should be transferred to liquid Nitrogen for long-term storage.
17. Unfortunately, 96-well plates can only be kept at -70°C. We do not recommend keeping the plates for more than 2 mo at this temperature. Therefore, all screening for the required alleles needs to be performed within this time period.
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