Team:USTC CHINA/Notebook/Protocols/Extracting plasmids from gram-negative bacterium

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<h1>Gel Extraction</h1>
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<h1>Extracting plasmids from gram-negative bacterium</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>1.Centrifuge 2-3ml bacteria liquid with 8000rpm and remove the supernatant; resuspense the liquid with lysyme(10mg/ml)~200ul and then leave it at temperature 37℃ for 0.5-1 hour.</br>
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Protocol
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2.Centrifuge the liquid with 8000rpm and remove the supernatant; resuspense it with 250uL ddH<sub>2</sub>O.</br>
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1. Excise gel slice containing DNA fragment of interest.
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3.Add 250uL buffer P2 (Alkaline lysis buffer), make it clear (reverse the tube evenly for several times) and leave it at room temperature for 2-4 minutes.</br>
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2. Add 3×sample volume of Buffer DE-A.
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4.Add 800uL Phenol-chloroform(PH8) and reverse the tube evenly until it layers.</br>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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5.Centrifuge the tube for 10 minutes and extract plasmid extraction column from the upper water phase.</br>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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6.The following steps are identical with those of Escherichia coli.</br>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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Repeat wash with Buffer W2
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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Latest revision as of 09:01, 27 September 2013

Extracting plasmids from gram-negative bacterium

1.Centrifuge 2-3ml bacteria liquid with 8000rpm and remove the supernatant; resuspense the liquid with lysyme(10mg/ml)~200ul and then leave it at temperature 37℃ for 0.5-1 hour.
2.Centrifuge the liquid with 8000rpm and remove the supernatant; resuspense it with 250uL ddH2O.
3.Add 250uL buffer P2 (Alkaline lysis buffer), make it clear (reverse the tube evenly for several times) and leave it at room temperature for 2-4 minutes.
4.Add 800uL Phenol-chloroform(PH8) and reverse the tube evenly until it layers.
5.Centrifuge the tube for 10 minutes and extract plasmid extraction column from the upper water phase.
6.The following steps are identical with those of Escherichia coli.

Notebook

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