Difference between revisions of "Team:UiOslo Norway/Experiments/Solubility Assay"

 
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<h3> Cell lysis using glass beads </h3>
 
<p>
 
<p>
 
<ul>
 
<ul>
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<li><p>Resuspend cell pellet in 600 ul PBS buffer (pH 7.4) </p></li>
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<li><p>Resuspend cell pellet in 600 µl PBS buffer (pH 7.4) </p></li>
 
<li><p>Add glass beads and disrupt cells by vortexing</p></li>
 
<li><p>Add glass beads and disrupt cells by vortexing</p></li>
 
<li><p>Centrifuge at 16.000 x g for 5 minutes</p></li>
 
<li><p>Centrifuge at 16.000 x g for 5 minutes</p></li>
 
<li><p>Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)</p></li>
 
<li><p>Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)</p></li>
 
</p>
 
</p>
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</ul>
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<h3> Cell lysis using sonication </h3>
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<p>
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<ul>
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<li><p>Resuspend the cell pellet in precooled imidazolebuffer. </p></li>
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<li><p>Sonicate 3x for 30 sec at a amplitude of 60. Incubate on ice for 1 minute between each sanitation step.</p></li>
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<li><p>Centrifuge the cell lysate at 13.000 rpm for 30 minutes at 4 °C.</p></li>
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<li><p>Keep the supernatant for further experiments</p></li>
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</p>
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<p><b>Imidazole buffer</b></br>
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</br>
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50 mM Tris-HCl pH 8 </br>
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100 mM NaCl</br>
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10 mM  beta-Mercaptoethanol</br>
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10 mM        Imidazole </br>
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<!---------------sponsors------------------------->
 
<!---------------sponsors------------------------->
  
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<center><h1>iGEM UiOslo 2015 was sponsored by:</h1>
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<center><h1>iGEM UiOslo 2015 is sponsored by:</h1>
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<hr style="height:2px;border:none;color:black; background-color:black;" />
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<hr style="height:2px;border:none;color:black;background-color:black;" />
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<img src="https://static.igem.org/mediawiki/2015/a/ac/UiOslo_Statoil.jpg" Height="60px" hspace="15">
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<img src="https://static.igem.org/mediawiki/2015/e/e0/UiOslo_uio_logo.png" Height="60px" hspace="15">
 
<img src="https://static.igem.org/mediawiki/2015/0/08/UiOslo_Enova1.jpg" Height="60px" hspace="15">
 
<img src="https://static.igem.org/mediawiki/2015/0/08/UiOslo_Enova1.jpg" Height="60px" hspace="15">
 
<img src="https://static.igem.org/mediawiki/2015/e/eb/UiOslo_GATC-Biotech.jpg" Height="60px" hspace="15">
 
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<img src="https://static.igem.org/mediawiki/2015/d/d9/UiOslo_Norwegian_Biochemical_Society.jpg" Height="60px" hspace="15">
 
<img src="https://static.igem.org/mediawiki/2015/d/d9/UiOslo_Norwegian_Biochemical_Society.jpg" Height="60px" hspace="15">
 
<img src="https://static.igem.org/mediawiki/2015/f/f0/UiOslo_SnapGene.jpg" Height="60px" hspace="15">
 
<img src="https://static.igem.org/mediawiki/2015/f/f0/UiOslo_SnapGene.jpg" Height="60px" hspace="15">
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<!--------end sponsors---------------------------->
 
<!--------end sponsors---------------------------->

Latest revision as of 12:19, 18 September 2015

Solubility Assay:

Back to Protocols


Cell lysis using glass beads

  • Resuspend cell pellet in 600 µl PBS buffer (pH 7.4)

  • Add glass beads and disrupt cells by vortexing

  • Centrifuge at 16.000 x g for 5 minutes

  • Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)

Cell lysis using sonication

  • Resuspend the cell pellet in precooled imidazolebuffer.

  • Sonicate 3x for 30 sec at a amplitude of 60. Incubate on ice for 1 minute between each sanitation step.

  • Centrifuge the cell lysate at 13.000 rpm for 30 minutes at 4 °C.

  • Keep the supernatant for further experiments

    • Imidazole buffer

    50 mM Tris-HCl pH 8
    100 mM NaCl
    10 mM beta-Mercaptoethanol
    10 mM Imidazole

    iGEM UiOslo 2015 is sponsored by: