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實驗課規範

實驗課規範. 請準時上課 請預讀實驗講義 避免因步驟繁雜而做不出實驗 請穿著 實驗衣 每天下課時先清點微量分注器 每天由各組排值日生幫忙清理實驗室. 報告 (8/10 統一由班代收齊繳交 ) 實驗報告 (50%) 目的 , 實際操作 步驟 , 結果 ( 包含判讀 ) 及討論 簡答與本週實驗相關問題共十題 (50%) 星期四早上隨堂考 ( 內容為星期一至星期三的上課及實驗內容 , 共 6 題 ) 星期五公佈星期四及星期五的題目 ( 共 4 題 ). Molecular cloning. 960709.

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實驗課規範

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  1. 實驗課規範 • 請準時上課 • 請預讀實驗講義 • 避免因步驟繁雜而做不出實驗 • 請穿著實驗衣 • 每天下課時先清點微量分注器 • 每天由各組排值日生幫忙清理實驗室

  2. 報告(8/10統一由班代收齊繳交) • 實驗報告(50%) • 目的,實際操作步驟,結果(包含判讀)及討論 • 簡答與本週實驗相關問題共十題(50%) • 星期四早上隨堂考(內容為星期一至星期三的上課及實驗內容,共6題) • 星期五公佈星期四及星期五的題目(共4題)

  3. Molecular cloning 960709

  4. Streaking E. coli on plate for competent cell production

  5. In vitro transcription Multiple cloning sites Expression cassette Selectable marker in prokaryotic cells Origen of replication (ORI) Selectable marker in eukaryotic cells

  6. pPDX1-FGF10 plasmid DNA

  7. 1 kb DNA marker

  8. pPDX1-FGF10 pcDNA3 Uncut plasmid DNA EcoRI-digested plasmid DNA pcDNA3 pPDX1-FGF10

  9. CIP and ligation

  10. The reaction catalyzed by DNA ligase (DNA replication: lagging strand)

  11. p.9

  12. p.7 65℃, 20min 37℃, 1hr Gel extraction

  13. Gel extraction/band isolation; p.11

  14. Uncut pcDNA3 EcoRI Gel extraction

  15. OD260 x 100 (dilution) x 50 mg/ml DNA Amount.= 1000 Determination of the amount of isolated DNA • Measure DNA concentration by UV spectrophotometer (分光光度計) • OD260=1 50 mg/ml • OD260/280=1.8~2.2 • 3 ml plasmid DNA + 297 ml ddH2O (100X dilution)

  16. Calculating the molar concentration (molarity) of vector and insert DNA Molar concentration (M) = [(mg/ml) ÷ (base pairs X 650 daltons)] X 2 ends Example: *Calculate the molarity of ends if you put 50 ng of a 5 kb vector in a 20 ml ligation reaction: 50 ng ÷ 20 μl = 0.0025 mg/ml [(0.0025 mg/ml) ÷ (5000 X 650)] X 2 = 1.54 X 10-9 M *Determine the amount of a 1 kb insert should be added to achieve a 1:6 vector:insert ratio: 6 X 1.54 X 10-9 M = 9.24 X 10-9 M [( ? mg/ml) ÷ (1000 X 650)] X 2 = 9.24 X 10-9 M ?= 0.003

  17. Actual Vector amount(mg/ml) X x ml = 0.0025 mg/ml X 20 ml • Actual insert amount (mg/ml) X y ml = 0.003 mg/ml X 20 ml = 0.06 mg

  18. Ligation V:I = 1:0 or 1:6(助教做) EcoRI/CIP-treated vector EcoRI-treated vector (Un-CIP) Vector (50 ng) x ul x ul Insert -- or y ul -- ul 10X buffer A 2 ul 2 ul 10X buffer B 2 ul 2 ul T4 DNA ligase (YEA) 1 ul 1 ul H2O add to 20 ul add to 20 ul  22oC (at D437), 1 hr  4oC overnight

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