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

Introduction molecular Biotech DNA RFLP (Southern) Hybridization Sequencing PCR

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

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

如何從檢體中獲得 DNA Whole blood

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

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

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

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

Polymerase chain reaction (PCR)

Restriction enzyme

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

Synthesis second-strand cDNA mRNA ss cDNA dsDNA PCR

Other Experiment Cell culture Western Blotting

Gains from learning Patience More attention Friendship

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