Molecular cloning 960709 Streaking E coli on plate

Molecular cloning 960709

Streaking E. coli on plate for competent cell production

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

p. PDX 1 -FGF 10 plasmid DNA

1 kb DNA marker

Uncut plasmid DNA pc. DNA 3 p. PDX 1 -FGF 10 Eco. RI-digested plasmid DNA pc. DNA 3 p. PDX 1 -FGF 10

CIP and ligation

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

p. 9

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

Gel extraction/band isolation; p. 11

Uncut pc. DNA 3 Eco. RI Gel extraction

Determination of the amount of isolated DNA • Measure DNA concentration by UV spectrophotometer (分光光度計) • OD 260=1 50 mg/ml • OD 260/280=1. 8~2. 2 • 3 ml plasmid DNA + 297 ml dd. H 2 O (100 X dilution) OD 260 x 100 (dilution) x 50 DNA Amount. = 1000 mg/ml

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

• 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

Ligation V: I = 1: 0 or 1: 6 Eco. RI/CIP-treated vector Vector (50 ng) x ul Insert -- or y ul 10 X buffer A 2 ul 10 X buffer B 2 ul T 4 DNA ligase (YEA) 1 ul H 2 O add to 20 ul 22 o. C (at D 437), 1 hr 4 o. C overnight (助教做) Eco. RI-treated vector (Un-CIP) x ul -- ul 2 ul 1 ul add to 20 ul