Team:Warsaw/Calendar-Main/29 July 2008

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<p>1. Isolation of plasmids from cultures inocluated on previous day.<br>
 
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2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains). 
 
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</p>
 
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<h3>Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha</h3>
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<ol><li>DNA fragment of omega_A isolated from gel on 20 July <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digestion</a> with SacI and NotI.</li>
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>clean-up</a>Clean-up of digested DNA fragment.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digestion</a> (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha. </li>
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<p><A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z_alpha</A> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2Bhis%2BZ%2Bomega>pET15b+Z_omega</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain inoculated in liquid LB with chloramphenicol and ampicillin.
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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 4300 bp band. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments from 2. and 4. (1 hr)</li>
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<h3>Cloning of omega_&Delta;A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a></h3><h4>Michał K.</h4>
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<ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a> from culture inoculated on previous day.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a>  with SacI and NotI (BamHI buffer).</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> (SacI and NotI) and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP) of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. </li>
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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 4300 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>) and 550 bp (omega_&Delta;A) bands (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_July_2008#fig1">Fig. 1.</a>).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments from 2. and 3. (1 hr).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation.</li>
<li>Transformants plating on LB + kanamycin.</li></ol>  
<li>Transformants plating on LB + kanamycin.</li></ol>  
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/6a/Po_go.jpg" width=200/></a> <var><b>Fig. 1. </b> SacI/NotI digests of isolated plasmids<br>
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1. Marker<br>
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2. digested pKS_omega_&Delta;A<br>
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3. digested pACYC_Omp_alpha<br></var>
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<h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance<br>
 
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Piotr</h3>
 
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<p>Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.</p>
 
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<center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
 
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<table id="result" align="center">
 
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<tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr>
 
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<tr><th>0</th><th>0.1</th><th>0.25</th><th>0.5</th><th>0.75</th><th>1</th></tr>
 
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<tr><th>25</th><td class="live">1.558</td><td class="live">1.469</td><td class="live">1.587</td><td class="live">1.49</td><td class="live">1.566</td><td class="live">1.311</td></tr>
 
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<tr><th>50</th><td class="live">1.425</td><td class="live">1.435</td><td class="live">1.524</td><td class="live">1.055</td><td class="live">0.920</td><td class="live">0.935</td></tr>
 
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<tr><th>75</th><td class="live">1.09</td><td class="live">0.989</td><td class="live">1.447</td><td class="live">0.971</td><td class="live">0.951</td><td class="live">0.992</td></tr>
 
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<tr><th>100</th><td class="live">0.09</td><td class="live">0.685</td><td class="live">1.378</td><td class="live">1.078</td><td class="live">0.977</td><td class="live">0.992</td></tr>
 
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</table>
 
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<p>
 
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Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)</p>
 
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<h3>Checking OmpA_omega_A_alpha and OmpA_A_alpha expression<br>
 
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Piotr</h3>
 
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<ol>
 
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<li>Spinning</li>
 
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<li>Suspending</li>
 
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<li>Adding of lysis buffer</li>
 
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<li>Boiling</li>
 
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<li>Putting into poliacrylamide gel</li>
 
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<li>Transfer onto nitrocellulose</li>
 
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<li>Blocking</li>
 
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<li>Anti-A antibody binding</li>
 
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<li>Washing</li>
 
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<li>Anti-rabbit antibody binding</li>
 
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<li>Developing with BCIP and NBT</li>
 
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</ol>
 
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[a photo of the gel is top be put here]
 
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<h4>Michał L., Ewa, Marcin</h4>
 
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<p>
 
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<ol>
 
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<li>Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin. </li>
 
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<li>Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.</li></ol>
 
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</p>
 
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Latest revision as of 16:55, 28 October 2008

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Purification of proteins: Z-alpha and Z-omega

Piotr

pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain inoculated in liquid LB with chloramphenicol and ampicillin.

Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

  1. Isolation of pKS+omega_ΔA from culture inoculated on previous day.
  2. Digest of pKS+omega_ΔA with SacI and NotI (BamHI buffer).
  3. Digest (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
  4. Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_ΔA) bands (Fig. 1.).
  5. Ligation of DNA fragments from 2. and 3. (1 hr).
  6. Transformation of E. coli TOP10 strain with ligation.
  7. Transformants plating on LB + kanamycin.

Fig. 1. SacI/NotI digests of isolated plasmids
1. Marker
2. digested pKS_omega_ΔA
3. digested pACYC_Omp_alpha

Retrieved from "http://2008.igem.org/Team:Warsaw/Calendar-Main/29_July_2008"