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Overview of Timeline for ES Cell Targeting and Southern Screening Thaw ES cell frozen stock and culture 5 days Electroporation of ES cells 24 hours Start Drug Selection 8-10 days Pick Colonies into 24-well plates 4-5 days Freeze colonies into 96 well plates and start making genomic DNA from the 24 well plates 5 days ES cells are lysed; DNA is precipitated and resuspended in TE 24 hours DNA is given to investigator to screen via Southern analysis for first positive conformation 2 weeks Investigator identifies positives and notifies TG facility of these 8 days TG facility expands clones from 96 well plate to a T25, freezes the clones, and gives investigator DNA from these clones for second positive confirmation 2 weeks Investigator re-confirms positive clones and notifies TG facility TG facility can begin blastocyst microinjections of modified ES cells, or can perform additional electroporations on these cell lines if needed Total estimated time: 35 days This does not include time taken by an investigator’s lab to perform Southern analysis for conformation of positive clones Also, due to the growth rate of cells, and differences in drug selection times, this timeline may be shortened or lengthened a bit