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Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates 1. Refeed cells 2-3 hours before use. 2. Check each well for confluence and number of clones. Selectively passage clones which have less than 10 colonies per well to another multiwell plate. 3. Aspirate off all of the media and wash 2 times with PBS. 4. Using the multi-channel pipetter, add 50 ul of trypsin to each of the wells. Change pipette tips between wells! 5. Incubate @ 37o C for 10-15 minutes. 6. Add 50 ul of 2X Freezing Media (60% DMEM, 20% FCS, 20% DMSO) to each well and break up the colonies by pipetting up-and-down about 5 times. 7. If required, remove 25 ul of the cell suspension and pipette directly into a new multiwell plate. If the cells are being grown up for Southern analysis, use a pre-gelatinized plate containing 200 ul of fresh M15 media per well. Alternatively, if the clones are being screened using PCR, use a 96-well plate containing PCR Lysis Buffer (50 ul + Proteinase K), lysing the cells either individually or in appropriately mixed pools. 8. Using the multi-channel pipetter, add 100 ul of filter-sterilized (0.22 um) Light Paraffin Oil to each well. This prevents degassing and evaporation during storage @ -70o C. 9. Replace the lid on the plate and secure by completely sealing with tape around all the edges. Place the plate in a polystyrene cuvette box, cover with a polystyrene lid that fits, secure with tape, and freeze @ -70o C (the temperature optimally should drop approximately 1o C/minute). 10. For samples being screened by PCR, take the plate of cells in PCR Lysis Buffer (prepared in step 7, above) and place @ 55o C for 1 hour to overnight. Adequate steps must be taken to avoid evaporation (e.g., addition of oil to cover each well and/or incubation in a humidified chamber, such as a sealed plastic container with a wet sponge inside). After incubation, the lysates may be stored @ -20o C. However, prior to use for PCR, the Proteinase K must be inactivated by transferring the lysates to labelled 0.5 ml Eppendorf tubes and heating @ 94o C for 15 minutes (Program #99 in the PCR machine). 11. To retrieve ES cell clones that have been frozen by this method, take the 96-well plate from the -70o C freezer and place directly into the 37o C incubator. Allow all of the wells to thaw completely (this may take 10-15 minutes for the wells near the center of the plate), then remove the clones from the wells and transfer to appropriately labelled wells in 24-well feeder plates pre-equilibrated with 2 ml of M15 media per well. For maximum recovery of sample, it is important to vigorously pipette the thawed cells to dislodge them from the bottom of the plate (where they settle during the freezing process). Since the cells tend to accumulate around the perimeter of the wells as a consequence of the minimal freezing volume, rinse the wells with media to further facilitate maximum recovery and transfer to the appropriate wells in the 24-well feeder plates. There is no need to remove the DMSO and the paraffin oil until the cells have replated (24 hours after passage). From the Laboratory of Dr. Allan Bradley Baylor College of Medicine, Houston, Texas