Team:Warsaw/Calendar-Main/22 July 2008

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<h3>Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA</h3>
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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>
<h4>Paweł</h4>
<p><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>
<p><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>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with NdeI and NotI (Orange buffer). Zero positive results obtained - just empty vectors.</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with NdeI and NotI (Orange buffer). Zero positive results obtained - just empty vectors.</li>
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<li>Another overnight <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation>ligation</a> of pET15b-OmpA-omega (NdeI/NotI cutted) with protein Z DNA.</li>
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<li>Another overnight <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation>ligation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a> (NdeI/NotI cut) with protein Z DNA.</li>
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<h3>Cloning of protein A DNA to OmpA constructs</h3><h4>Michał K.</h4>
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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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<ol><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 (pACYC177+OmpA_A_omega).</li>
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<h4>Michał L., Ewa, Marcin</h4>
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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. </li></ol>
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<p>
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We have obtained white colonies of transformants and inoculated 10 of them on liquid LB+Amp<sub>100</sub>.</p>
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<h3>Cloning omega-A fusion on pKS (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>Digest of plasmids with SacI and NotI (BamHI buffer).</li>
 
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<li>Gel electrophoresis of productsof 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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<h3>Cloning of protein A DNA to OmpA constructs</h3><h4>Michał K.</h4>
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<p><ol>
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<li>Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations <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-A-alpha>pACYC177+OmpA_A_alpha</a>. Primers used:
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI.</a>
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</li><li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/22_July_2008#fig1">Fig. 1.</a>).</li>
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<li> Confirmed transformant colonies (found only on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>pACYC177+OmpA_A_alpha</a> plate) inoculated to liquid LB with kanamycin. </li></ol>
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</p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/1/1f/Ompa_ompa.jpg" width=340/></a> <var><b>Fig. 1. </b>Colony PCR on transformants with pACYC177_OmpA_alpha_A<br>
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Upper and lower:
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1. Marker<br>
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2-13. colony PCR (pACYC177_OmpA_alpha_A)<br></var>

Latest revision as of 21:51, 27 October 2008

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

Paweł

  1. Isolation of plasmids from cultures inocluated on previous day.
  2. Control digest of isolated plasmids with NdeI and NotI (Orange buffer). Zero positive results obtained - just empty vectors.
  3. Another overnight ligation of pET15b-OmpA-omega (NdeI/NotI cut) with protein Z DNA.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

We have obtained white colonies of transformants and inoculated 10 of them on liquid LB+Amp100.

Cloning of protein A DNA to OmpA constructs

Michał K.

  1. Colony PCR on colonies from plates with transformations pACYC177+OmpA_A_omega and pACYC177+OmpA_A_alpha. Primers used: AL+SacI and AP+NotI.
  2. Gel electrophoresis (Fig. 1.).
  3. Confirmed transformant colonies (found only on pACYC177+OmpA_A_alpha plate) inoculated to liquid LB with kanamycin.

Fig. 1. Colony PCR on transformants with pACYC177_OmpA_alpha_A
Upper and lower: 1. Marker
2-13. colony PCR (pACYC177_OmpA_alpha_A)

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