Team:USTC CHINA/Notebook/Protocols/Sample analysis

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<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
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<h1>Gel Extraction</h1>
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<h1>Sample analysis</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>
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Protocol
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<span>1. Preparation of soluble and insoluble cell extracts from B. subtilis</span></br>
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1. Excise gel slice containing DNA fragment of interest.
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- harvest cells by centrifugation (10 min, 6,000 x g, 4 °C)</br>
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2. Add 3×sample volume of Buffer DE-A.
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- wash and resuspend in 50 mM sodium phosphate buffer (pH 7.0) at an OD600 of 10</br>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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- disrupt cells by ultrasonication (12 W, 6 x 15 pulses with 15 sec intervals) in 1.5 ml</br>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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Eppendorf tubes containing 1 ml of cell suspension, supplemented with lysozyme</br>
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<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
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(250 μg/μl, CB-0663-5GAM), on ice.</br>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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- alternatively, cells can be disrupted by beat beating:</br>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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disrupt three times with glass beads (0.1 mm in diameter) (1 g/ml of cell suspension) in</br>
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Repeat wash with Buffer W2
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an orbital mixer at 180 V, with the mix kept on ice for 3 min between each disruption</br>
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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- take 100 μl of the preparation as first total protein sample (T1)</br>
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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- remove cell debris by centrifugation at 4,300 x g, 10 min</br>
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- take 100 μl of the supernatant for the second total protein sample (T2)</br>
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- spin at 8.200 x g (10 min, 4 °C) </br>
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to separate into insoluble (I) and soluble (S) protein fractions.</br>
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- per sample use the amount of protein corresponding to 0.025 of OD600 for separation by SDS-PAGE</br>
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- analyze samples by immunoblotting with specific antiserum</br>
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Latest revision as of 09:12, 27 September 2013

Sample analysis

1. Preparation of soluble and insoluble cell extracts from B. subtilis
- harvest cells by centrifugation (10 min, 6,000 x g, 4 °C)
- wash and resuspend in 50 mM sodium phosphate buffer (pH 7.0) at an OD600 of 10
- disrupt cells by ultrasonication (12 W, 6 x 15 pulses with 15 sec intervals) in 1.5 ml
Eppendorf tubes containing 1 ml of cell suspension, supplemented with lysozyme
(250 μg/μl, CB-0663-5GAM), on ice.
- alternatively, cells can be disrupted by beat beating:
disrupt three times with glass beads (0.1 mm in diameter) (1 g/ml of cell suspension) in
an orbital mixer at 180 V, with the mix kept on ice for 3 min between each disruption
- take 100 μl of the preparation as first total protein sample (T1)
- remove cell debris by centrifugation at 4,300 x g, 10 min
- take 100 μl of the supernatant for the second total protein sample (T2)
- spin at 8.200 x g (10 min, 4 °C)
to separate into insoluble (I) and soluble (S) protein fractions.
- per sample use the amount of protein corresponding to 0.025 of OD600 for separation by SDS-PAGE
- analyze samples by immunoblotting with specific antiserum