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Analysis of ES Cell Clones by Mini-Southern Initial Cloning 1. Aspirate selective media off the plates containing colonies of interest. Wash the plates twice with PBS. After aspirating the second wash, add enough PBS to cover the surface of the dish. 2. Prepare the 96-well U-bottom cloning dish by pipetting 25 ul of trypsin into each well using a multichannel pipettor. Up to two full plates of clones can be picked at one sitting without problems of the trypsin drying. 3. Pick colonies from the washed plates and transfer them into the trypsin solution, one colony per well. Proceed until colonies have been cloned into each of the 96 wells. Approximately one hour will have elapsed by the end of the process (assuming a picking speed of 100-150 colonies/hour), enough time to ensure complete trypsinization of the colonies. Therefore, no further incubation is necessary. 4. Using the multichannel pipettor, add 25 ul of M15 media per well. Pipette up-and-down several times to disaggregate the cells. 5. Transfer the clones to an appropriately labelled 96-well feeder plate and allow the colonies to grow under selection. The cells will take about 3-5 days to grow to such a confluence as to turn the media yellow at one-day intervals. Duplicate Preparation 1. Once the clones reach confluence, trypsinize, duplicate, and freeze the cells [refer to the protocol "Freezing Embryonic Stem ES) Cell Clones in 96-Well Plates"]. 2. Allow the cells to grow on the gelatinized 96-well duplicate plates. Refeed the plates with fresh M15 media as the old media turns yellow until the cells are ready for use. Mini-Southern Analysis 1. When the cells are ready, wash the plates twice with PBS and aspirate. Using the multichannel pipettor, add 50 ul of Lysis Buffer [10 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 10 mM NaCl, 0.5% Sarcosyl, and 1 mg/ml Proteinase K (added fresh)] per well. Note: From this point on, all manipulations are to be performed outside the tissue culture facilities. 2. Incubate the plates overnight @ 60o C in a humidified chamber (such as a plastic container with a wet sponge on the inside). 3. Next day, prepare a fresh solution of 75 mM NaCl in ethanol (add 150 ul of 5 M NaCl per 10 ml of cold absolute ethanol and mix well; the salt will precipitate, but this is of little consequence). 4. Using the multichannel pipettor, add 100 ul of the NaCl/ethanol solution per well. Allow the plate to rest on the bench @ room temperature for 15-30 minutes, or until the precipitated DNA is clearly visible under low-power magnification. The DNA adheres to the plastic, so look at the perimeter of each well to see the precipitated DNA. 5. Invert the plate to discard the solution (the DNA will remain adhered to the plate). Using the multichannel pipettor, add 70% ethanol to wash each well. Alternatively, a squirt bottle may be used, but a strong stream could detach the DNA from the plate. Invert the plate to discard the 70% ethanol and repeat the wash 2-3 times. 6. After the final wash, invert the plate, discard the 70% ethanol, and allow the plate to air-dry a few minutes. 7. While the plate is drying, prepare the Restriction Enzyme Cocktail (1X Restriction Buffer specified for the enzyme being used, 1 mM Spermidine, 100 ug/ml Bovine Serum Albumin, and 10-15 units of enzyme). 8. Using the multichannel pipettor, add 30 ul of Restriction Enzyme Cocktail to each well and mix by pipetting up-and-down. Change tips between one row and the next. 9. Once the cocktail has been added to all the wells, incubate the plates overnight @ 37o C in a humidified chamber. 10. Next day, prepare the agarose gel(s) for electrophoresis. Prepare a large gel tray (15 cm x 24 cm) with three 33-teeth combs (add 3 extra teeth per comb, secured with tape) evenly distributed along the length of the tray. Pour 300 ml of molten agarose into the tray; remove any bubbles with a needle. Allow the gel to solidify for about 30 minutes. This size of gel (3 lanes with 99 wells total) will accommodate one 96-well mini-Southern digest plate at 32 samples per lane plus one well per lane for molecular weight markers. 11. Remove the 96-well mini-Southern digest plate from the incubator and add 4-5 ul of loading buffer to each well. Load the gel (30-35 ul per well) and run @ 80 V for approximately 4 hours. When deciding how far to allow the samples to migrate, take into account the size of the fragment(s) being distinguished. The gel can be run further as long as the bands from one lane of samples being detected with a probe do not overlap the bands from the next lane of samples. 12. After the electrophoresis is complete, transfer the DNA to GeneScreen Plus membranes according to the usual protocol [refer to the protocol "Genomic DNA: Restriction Enzyme Digests, Agarose Gel Electrophoresis, and Southern Transfer (Blotting)"]. NOTES: This protocol may not work efficiently for all restriction enzymes. Therefore, make a pilot experiment to test the desired enzyme(s) before proceeding to the large experiment. Before transferring the DNA to membranes, discard the areas of the agarose gel that are not needed for probing. From the Laboratory of Dr. Allan Bradley Baylor College of Medicine, Houston, Texas