Team:Warsaw/Calendar-Main/2 October 2008

From 2008.igem.org

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<h3>Preparation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _alpha</h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<p> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009)</a>.</p>
<p> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103009>linker_alpha (BBa_K103009)</a>.</p>
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<h3>Preparation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _omega</h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<p>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _omega.</p>
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<p>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a>.</p>
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<h3>Preparation of pACYC177 + OmpA_omega_</h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<p>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments isolated  on <a href=https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008>30 September</a> : pACYC177 + OmpA_omega_.</p>
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<p>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments isolated  on <a href=https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008>30 September</a> : <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a>.</p>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a></h3>
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<h3>Preparation of BioBricks</h3>
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<h4>Michał K.</h4>
<h4>Michał K.</h4>
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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 culture inoculated on previous day (<a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a>).</li>
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<ol><li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a>
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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 plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found.</li>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pLac_OmpA_omega fragment with EcoRI and BcuI (BamHI buffer). </li>
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<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 1200 bp. </li>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a>
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  primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+OmpA (annealing temperature - 55&deg;C,45 s of elongation step). </li>
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  primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a> (annealing temperature - 55&deg;C,45 s of elongation step). </li>
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<li> Gel electrophoresis.</li>
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<li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/2_October_2008#fig1">Fig. 1</a>).</li>
<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin.</li>
<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin.</li>
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</ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/9/92/Kolonijny_rfp_omp_03_10_2008.jpg"></a>
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<var><b>Fig. 1.</b>Colony PCR with OmpaL_N and OmpaP_link. <br>
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1. Marker<br>
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4, 14, 19, 20, 24. Proper bands visible. <br> </var>
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</ol>
 
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<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a></h3>
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<h4>Michał K.</h4>
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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 culture inoculated on previous day (<a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</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 plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found (<li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/2_October_2008#fig2">Fig. 2</a>).</li></ol>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/6/6f/Traw_02_10_2008.jpg"></a>
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<var><b>Fig. 2.</b>Control digest of pSB1A3+Z(BBa_K103004)<br>
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1. Marker<br>
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3-5. Clones carrying BBa_K103004</var>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a></h3>
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<h4>Michał K.</h4>
 +
<ol>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> fragment with EcoRI and BcuI (BamHI buffer). </li>
 +
<li> Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 1200 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/2_October_2008#fig3">Fig. 3</a>.</li>
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 +
</ol>
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<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/5/54/Go_02_10_2008_na_2_10.jpg"></a>
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<var><b>Fig. 3.</b>Digest of OmpA_linker_omega_linker under Plac (BBa_K103018).<br>
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1. Marker<br>
 +
2. OmpA_linker_omega_linker under Plac (BBa_K103018)<br></var>

Latest revision as of 20:34, 28 October 2008

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Preparation of linker_alpha (BBa_K103009)

Michał K.

Overnight ligation of isolated DNA fragments: pSB2K3 + linker_alpha (BBa_K103009).

Preparation of linker_omega (BBa_K103013)

Michał K.

Overnight ligation of isolated DNA fragments: pSB2K3 + linker_omega (BBa_K103013).

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

Overnight ligation of DNA fragments isolated on 30 September : pACYC177 + OmpA-linker-omega-linker (BBa_K103016).

Preparation of OmpA-linker (BBa_K103006)

Michał K.

  1. Colony PCR with OmpaL_N and OmpaP_link primers on colonies from plates with transformations pSB1A3+OmpA-linker (BBa_K103006) (annealing temperature - 55°C,45 s of elongation step).
  2. Gel electrophoresis (Fig. 1).
  3. Confirmed transformant colonies inoculated to liquid LB with ampicillin.
Fig. 1.Colony PCR with OmpaL_N and OmpaP_link.
1. Marker
4, 14, 19, 20, 24. Proper bands visible.

Preparation of Z(BBa_K103004)

Michał K.

  1. Isolation of plasmids from culture inoculated on previous day (pSB1A3+Z(BBa_K103004)).
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found (
  3. Gel electrophoresis (Fig. 2).
Fig. 2.Control digest of pSB1A3+Z(BBa_K103004)
1. Marker
3-5. Clones carrying BBa_K103004

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

  1. Digest of BBa_K103018 fragment with EcoRI and BcuI (BamHI buffer).
  2. Gel electrophoresis and gel-out of proper band - 1200 bp. Fig. 3.
Fig. 3.Digest of OmpA_linker_omega_linker under Plac (BBa_K103018).
1. Marker
2. OmpA_linker_omega_linker under Plac (BBa_K103018)

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