Team:Warsaw/Calendar-Main/14 August 2008

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<h3>Cloning of alpha to <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day. </li>
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<ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of plasmids from cultures inocluated on previous day. </li>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and NotI (BamHI buffer).  
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of isolated plasmids with SacI (SacI buffer), vectors were also <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylated</a> with CIAP.  
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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>
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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 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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<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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1. Purified Z-omega<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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4. Purified Z-alpha<br>
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5. Marker<br></var>
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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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1. Marker<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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Latest revision as of 15:40, 28 October 2008

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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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