Summer Research The Molecular Mechanisms of Breast Tumorigenesis

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

Introduction molecular Biotech DNA n RFLP n (Southern) Hybridization n Sequencing n PCR

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

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

如何從檢體中獲得DNA n Whole blood

Process n Centrifuge n Add RBC lysis buffer n Add WBC lysis buffer n 550 C over night n Phenol/chloroform extraction n Salt out

Process n 100%Et. OH n Dissolve DNA n Test OD n PCR n AGAROSE n Film(result)

DNA extracted from DNA/protein mixture Phenol/chloroform Phenol/ chloroform centrifugation liquid phase (DNA) organic phase (protein etc. ) WBC & protein mixture @ 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

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

Polymerase chain reaction (PCR)

Restriction enzyme

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

Synthesis second-strand c. DNA m. RNA ss c. DNA ds. DNA PCR

Other Experiment n Cell culture n Western Blotting

Gains from learning Patience More attention Friendship

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