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

Streaking E. coli on plate for competent cell production

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

pPDX1-FGF10 plasmid DNA

1 kb DNA marker

Uncut plasmid DNA pcDNA3 pPDX1-FGF10 EcoRI-digested plasmid DNA pcDNA3pPDX1-FGF10

CIP and ligation

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

p.9

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

Gel extraction/band isolation; p.11

Gel extraction EcoRI Uncut pcDNA3

Determination of the amount of isolated DNA Measure DNA concentration by UV spectrophotometer ( 分光光度計 ) OD 260 =1  50  g/ml OD 260/280 =1.8~2.2 3  l plasmid DNA  l ddH2O (100X dilution) OD 260 x 100 (dilution) x  g/  l DNA Amount.=

Calculating the molar concentration (molarity) of vector and insert DNA Molar concentration (M) = [(  g/  l) ÷ (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  l ligation reaction: 50 ng ÷ 20 μl =  g/  l [(  g/  l) ÷ (5000 X 650)] X 2 = 1.54 X 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 M = 9.24 X M [( ?  g/  l) ÷ (1000 X 650)] X 2 = 9.24 X M ?= 0.003

Actual Vector amount (  g/  l) X x  l =  g/  l X 20  l Actual insert amount (  g/  l) X y  l =  g/  l X 20  l = 0.06  g

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  22 o C (at D437), 1 hr  4 o C overnight