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Monday, Day 13
View the 10 cm dishes containing the transfected ES cells through the inverted microscope. The clones are visible as small nests of rapidly growing cells. They have tight borders and are closely packed. Larger cells within the colony, with well defined membranes are the cells which are beginning to differentiate. Do not pick these clones! This is day 6 of the selection process. If the clones are big enough you may pick on this day, or wait until day 7. To pick the clones, you must view them through an inverted microscope in a laminar flow hood. Prepare your 24 well plates (made on Friday, Day 10) by taking out the old media and replacing it with 1.0 ml ES Selection Media per well. Place the 24 well dishes (all 6) back into the incubator. Replace the ES selection media in the first 10 cm dish with fresh ES Selection Media. Place the dish under the microscope and view the cells at 4X and 10X trying to be sure you are picking clones that have not yet started to differentiate. Isolate the clone that you wish to pick in the viewing field. Using a 0-160 ul barrier tip, gently push the ES clone forward from the surrounding MEFs. With the pipettor set between 30 and 50 ul, and the plunger button already depressed, pluck the clone using a forward scooping suction motion. If the pipettor is set on 30 ul, you should have enough suction to dislodge the clone from the plate. However, if the inactivated MEF layer is too dense, you may have trouble dislodging the ES clones. In this case, you must carefully tease the surrounding MEFs away from the clone without disrupting the clone with your tip. Then you should be able to harvest your clones as above. Place each clone into one of the 24 wells containing ES Selection Media. Continue to pick clones and place in the wells of the prepared 24 well plate until 12 clones are picked or the 10 cm. dish has been out of the incubator for 15 minutes (the clones are sensitive to pH and temperature changes). Now place the plate containing the picked clones in the incubator. Continue picking clones until all 24 wells have been filled in all of your prepared dishes. Leave the 24 well dishes in the incubator overnight.
Tuesday, Day 14
If you were not able to pick all your clones on day 13, you may continue to pick today (selection day 7). We do not recommend picking on day 8.
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