Team:Warsaw/Calendar-Main/14 August 2008

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<li> Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands 4400 bp (for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a>) and 4200 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a>). </li></ol>
<li> Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands 4400 bp (for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a>) and 4200 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a>). </li></ol>
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
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<p>Electrophoresis in polyacrylamide gel (12 %) of sonicate and preliminarily purified protein fusios: Z-alpha and Z-omega (Fig. 1., Fig. 2.)</p><br>
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<p>Electrophoresis in polyacrylamide gel (12 %) of sonicate and preliminarily purified protein fusions: Z-alpha and Z-omega (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/14_August_2008#fig1">Fig. 1.</a> and <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/14_August_2008#fig2">Fig. 2.</a>)</p><br>
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<img src="https://static.igem.org/mediawiki/2008/3/35/August_14_th_2.jpg" width=250 /><var>Fig. 1. <b>Fractions from purifying Z-alpha and Z-omega protein fusions.<br>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/3/35/August_14_th_2.jpg" width=250 /></a><var><b>Fig. 1.</b> Fractions from purifying Z-alpha and Z-omega protein fusions.<br>
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</b><br>  
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<br>  
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1. purified Z-omega<br>
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1. Purified Z-omega<br>
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2. supernatant Z-alpha after sonication<br>
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2. Supernatant Z-alpha after sonication<br>
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3. elution of Z-alpha using ddH2o<br>
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3. Elution of Z-alpha using ddH2o<br>
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4. purified Z-alpha<br>
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4. Purified Z-alpha<br>
5. Marker<br></var>
5. Marker<br></var>
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<img src="https://static.igem.org/mediawiki/2008/5/5d/August_14_th.jpg" width=250 /><var>Fig. 2. <b>Sonicate and pellet from Z-omega induction<br>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/5/5d/August_14_th.jpg" width=220 /></a><var><b>Fig. 2.</b> Sonicate and pellet from Z-omega induction<br>
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</b><br>  
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<br>  
1. Marker<br>
1. Marker<br>
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2. sonicate Z-omega after induction<br>
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2. Sonicate Z-omega after induction<br>
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3. pellet Z-omega after induction<br></var>
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3. Pellet Z-omega after induction<br></var>

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Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Isolation of plasmids from cultures inocluated on previous day.
  2. Digest of isolated plasmids with SacI (SacI buffer), vectors were also dephosphorylated with CIAP.
  3. Gel electrophoresis and gel-out of proper bands 4400 bp (for pACYC177+OmpA_A_omega) and 4200 bp (pACYC177+OmpA_Z_omega).

Purification of proteins: Z-alpha and Z-omega

Piotr

Electrophoresis in polyacrylamide gel (12 %) of sonicate and preliminarily purified protein fusions: Z-alpha and Z-omega (Fig. 1. and Fig. 2.)


Fig. 1. Fractions from purifying Z-alpha and Z-omega protein fusions.

1. Purified Z-omega
2. Supernatant Z-alpha after sonication
3. Elution of Z-alpha using ddH2o
4. Purified Z-alpha
5. Marker
 Fig. 2. Sonicate and pellet from Z-omega induction

1. Marker
2. Sonicate Z-omega after induction
3. Pellet Z-omega after induction



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