1. 沈志陽 老師 The Molecular Mechanisms of Breast Tumorigenesis 生科 05 陳俊朝 2003/10/30 Summer Research

2. Introduction molecular Biotech DNA RFLP (Southern) Hybridization Sequencing PCR

3. Introduction molecular Biotech RNA d(Northern) hybridization, RT-PCR Microarray

4. Introduction molecular Biotech Protein Western blotting (immunoblotting) Immunohistochemical staining Functional assay

5. 如何從檢體中獲得 DNA Whole blood

6. Process Centrifuge Add RBC lysis buffer Add WBC lysis buffer 55 0 C over night Phenol/chloroform extraction Salt out

7. Process 100%EtOH Dissolve DNA Test OD PCR AGAROSE Film(result)

8. DNA extracted from DNA/protein mixture Phenol/chloroform WBC & protein mixture Phenol/ chloroform liquid phase (DNA) organic phase (protein etc.) centrifugation @ phenol : 溶解小蛋白質與 petide, 不溶 DNA, 水會溶於 phenol @ chloroform : lipid 破壞, protein denature 將 phenol 帶離水層

9. DNA precipitation (Salt out) DNA Sodium acetate or NH 4 OAc >2 vol EtOH centrifugation DNA @ Salt : DNA neutralization @ EtOH : DNA dehydration Dry out 1.store 4 o C or -20 o C 2.TE butter

10. Polymerase chain reaction (PCR)

11. Restriction enzyme

12. RNA extraction from blood RBC lysis Wash WBC lysis Homogenize with QIAshedder Add 70% ethanol Bind total RNA to RNeasy membrane wash 3x Elute Ready to use RNA

13. Synthesis second-strand cDNA mRNA ss cDNA dsDNA PCR

14. Other Experiment Cell culture Western Blotting

15. Gains from learning Patience More attention Friendship

16. Thank you for your listening 插滴 XD