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Milap Thaker - Biology Powerpoint: Harvard University - DNA Damage Checkpoints
1. Fengshan Liang and Yanchang Wang DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms MOLECULAR AND CELLULAR BIOLOGY, July 2007, p. 5067–5078 Presenter: MilapThaker Proseminar in Molecular and Cellular Biology Harvard University Date 11/29/2007
2. Key Point 1: Two Different Mechanisms Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 2
3. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 3 Key Point 1: Two Different Mechanisms
5. Relevant context: How to activate Mitotic Exit Network? Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 5
6. Mitotic Exit Network: Take 2 MilapThaker - Graduate Proseminar - Molecular and Cellular Biology 6
7. Alternate Mechanism: The FEAR Pathway:FEAR: CDC14 Early Anaphase Release Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 7
8. Mitotic Exit Network vs. CDC14 Release: Specific Temporal Differences Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 8
9. Materials and Methods Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium Arrested in G1 by 5 µg/ml α-factor After 2 hours, washed 2x with water Cytology: Cells fixed with 3.7% formaldehyde for 5 min at RT, then washed twice with PBS buffer (phosphate buffered saline) and resuspended in PBS Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 9
10. DAPI Staining Technique Cells fixed with 100% MeOH at -20 Celsius for 30 mins. Cells pelleted, resuspended in DAPI at a final concentration of 2.5µg/ml for 1 min at RT Cells finally resuspended in 1X PBS Fluorescence microscopy used to examine GFP and DNA Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 10