Team:Warsaw/Calendar-Main/23 July 2008

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<h3>Cloning omega-A fusion on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a> (second attempt)</h3>
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<h4>Michał L., Ewa, Marcin</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 plasmids from transformants.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of plasmids with SacI and NotI (BamHI buffer).</li>
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<li>Gel electrophoresis of products of digest.</li>
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<li>2 selected clones were send to DNA sequencing lab.</li>
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</ol>
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<p>'''Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha''' <br>
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<h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a> in place of OmpA</h3><h4>Paweł</h4>
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1. DNA fragment of omega_A isolated from gel on 12 July digested with SacI and NotI. <br>
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<p>Ligations <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformed</a> into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> and plated on LB + ampicillin.</p>
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2. Clean-up of digested DNA fragment. <br>
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3. Digest (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha. <br>
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4. Gel electrophoresis and gel-out of 4300 bp band. <br>
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5. Ligation of DNA fragments from 2. and 4. (1 hr)<br>
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6. Transformation of E. coli TOP10 strain with ligation. <br>
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<h3>Preparation of chemocompetent bacteria</h3>
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7. Transformants plating on LB + kanamycin.  
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<h4>Antoni</h4>
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<p>
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Setup of overnight culture <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> (LB broth) for preparation of chemocompetent bacteria.</p>
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<h3>Cloning of protein A DNA to OmpA constructs</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 of plasmids</a> from cultures inocluated on previous day (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>pACYC177+OmpA_A_alpha</a>). </li>  
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<li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with BamHI and SacI (BamHI buffer).</li><li> Gel electrophoresis - proper clone found (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/23_July_2008#fig1">Fig. 1.</a>).</li></ol>
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<p>Paweł: ligation results: 15 colonies grown. All of them cultured overnight in LB+amp.
 
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/f/fb/23_july.jpg" width=300/></a> <var><b>Fig. 1. </b>Control SacI/BamHI digests of pACYC177+OmpA_A_alpha<br>
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1-4. digested plasmids isolated from transformants obtained previous day<br>
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5. Marker<br></var>
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</html>

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Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

  1. Isolation of plasmids from transformants.
  2. Digest of plasmids with SacI and NotI (BamHI buffer).
  3. Gel electrophoresis of products of digest.
  4. 2 selected clones were send to DNA sequencing lab.

Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA

Paweł

Ligations transformed into TOP10 and plated on LB + ampicillin.

Preparation of chemocompetent bacteria

Antoni

Setup of overnight culture E. coli TOP10 (LB broth) for preparation of chemocompetent bacteria.

Cloning of protein A DNA to OmpA constructs

Michał K.

  1. Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_A_alpha).
  2. Control digest of isolated plasmids with BamHI and SacI (BamHI buffer).
  3. Gel electrophoresis - proper clone found (Fig. 1.).

Fig. 1. Control SacI/BamHI digests of pACYC177+OmpA_A_alpha
1-4. digested plasmids isolated from transformants obtained previous day
5. Marker