Team:KAIST Korea/Notebook Protocol

From 2012.igem.org

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<h3>Procedure</h3></br>
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<span id="starter">P</span>rotocol 3 content<br /><br /><br /><br /><br /><br />
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<h4>1) Restriction enzyme digestion</h4>
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<li>Incubate 2hr at 37℃.</li>
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<li>Inactivation 20min at 65℃.</li>
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<h4>2) Dephosphorylation (Only for vector)</h4>
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<li>Incubate 30min at 37℃.</li>
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<li>Inactivation 5min at 70℃.</li>
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<h4>3) Ligation</h4>
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<li>Incubate 16hr at 16℃.</li>
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<h4>4) Transformation</h4>
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<li>Add 20ul ligated vector into 100ul of competent cell.</li>
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<li>Incubate ice 5min.</li>
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<li>Heat shock 42℃, 1min 30sec.</li>
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<li>Ice 5min.</li>
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<li>Recovery with 700ul LB at 37℃, 1hr.</li>
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<li>Plating.</li>
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Revision as of 11:07, 23 August 2012

KAIST Korea 2012 iGEM

image description

Lab Protocols

Please enter sub title


1. LB agar plate

Materials

materials


Procedure

  1. Add 25g LB broth and 15g agar into 1L DDW.
  2. Autoclave
2. Genomic DNA Purification

Materials

materials


Procedure


Pellet Cells

  1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
  2. Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.
  3. Remove the supernatant.

Lyse Cells

  1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
  2. Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.
  3. Remove the supernatant.

Protein Precipitation

  1. Add 200ul of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20seconds to mix the Protein Precipitation Solution tieh the cell lysate.
  2. Incubate the sample on ice for 5minutes.
  3. Centrifuge at 13,000rpm 3minutes.
  4. Transfer the supernatant containing the DNA to a clean 1.5ml microcentrifuge tube.
    5. Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.
  5. Add 600ul isopropanol.
  6. Gently mix by inversion until the thread-like strands of DNA form a visible mass.
  7. Centrifuge at 13000rpm for 2minutes.
  8. Carefully pour off the supernatant.
  9. Add 600ul of room temperature 70% ethanol and gently invert the tube 5 times to wash the DNA pellet.
  10. Centrifuge at 13000rpm for 2minutes. Carefully aspirate the ethanol.
  11. Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-14minutes.
  12. Add 50~100 μl TE buffer and rehydrate the DNA by incubating at 65℃ for 20min. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubationg the solution overnight at room temperature or at 4℃.

TE buffer
3. Vector transformation

Procedure


1) Restriction enzyme digestion

Restriction enzyme digestion
  1. Incubate 2hr at 37℃.
  2. Inactivation 20min at 65℃.

2) Dephosphorylation (Only for vector)

Dephosphorylation (Only for vector)
  1. Incubate 30min at 37℃.
  2. Inactivation 5min at 70℃.

3) Ligation

Ligation
  1. Incubate 16hr at 16℃.

4) Transformation

  1. Add 20ul ligated vector into 100ul of competent cell.
  2. Incubate ice 5min.
  3. Heat shock 42℃, 1min 30sec.
  4. Ice 5min.
  5. Recovery with 700ul LB at 37℃, 1hr.
  6. Plating.
Protocol 4 title
Protocol 4 content





Protocol 5 title
Protocol 5 content





Protocol 6 title
Protocol 6 content





Protocol 7 title
Protocol 7 content 아졸려 언제와 점심먹고 왔는데 아무도 없어





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