1. Working one row at a time using a multichannel pipetter, change the medium 23 h prior to freezing.
2. Freshly prepare 2X cell-freezing media.
3. Aspirate the medium from each well, and wash the cells with PBS (approx 200 |L).
4. Add 50 |L trypsin to each well, and then place plate in an incubator for 5-10 min.
5. Working on ice, preferably in a wide, flat container, aliquot 50 |L of DMEM+ into each well. Pipet the cells several times in order to get them into a homogenous suspension.
6. Then add 100 |L 2X cell freezing media to the wells, and again pipet to mix.
7. Finally add 80-100 ||L sterile mineral oil (Sigma, cat. no. M-8410) to cover the cell/freezing medium mixture.
8. Wrap the plates in parafilm, place in a styrofoam box, and store in a -70°C freezer until such time as the desired clones have been identified and need to be recovered (see Note 17).
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