Team:USTC CHINA/Notebook/Protocols/The transformation of E.coli

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<h1>Gel Extraction</h1>
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<h1>The transformation of E.coli</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. Remove competent cells (200μl/tube) from freezer and allow to thaw on ice for 3 min.</br>
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Protocol
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2. Add 3-5 μl of DNA to the cells.</br>
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1. Excise gel slice containing DNA fragment of interest.
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3. Incubate on ice for 30 min.</br>
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2. Add 3×sample volume of Buffer DE-A.
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4. Heat shock the cells at 42°C for 45 seconds.</br>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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5. Add 800 μl of LB Broth, and incubate in a shaker at 37°C for 45~60min.</br>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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6. Centrifuge at 12,000 for 1 min, discard 800 μl supernatant.</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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7. Re-suspend the pellet in the 200 μl of supernatant and spread onto one agar plate.</br>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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8. Incubate the plates overnight at 37°C.</br>
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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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Revision as of 17:17, 25 September 2013

The transformation of E.coli

1. Remove competent cells (200μl/tube) from freezer and allow to thaw on ice for 3 min.
2. Add 3-5 μl of DNA to the cells.
3. Incubate on ice for 30 min.
4. Heat shock the cells at 42°C for 45 seconds.
5. Add 800 μl of LB Broth, and incubate in a shaker at 37°C for 45~60min.
6. Centrifuge at 12,000 for 1 min, discard 800 μl supernatant.
7. Re-suspend the pellet in the 200 μl of supernatant and spread onto one agar plate.
8. Incubate the plates overnight at 37°C.

notebook

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