Team:Warsaw/Calendar-Main/9 October 2008

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<h3>Preparation of BioBricks</h3>
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<h3>Preparation of vector for pT7 constructs</h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol>
<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 cultures inoculated on previous day <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ AID and pACYC177 + <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI).</li>
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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 cultures inoculated on previous day - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> (with removed XbaI site).</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 <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ AID plasmids with EcoRI and PstI (Orange buffer) and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found for both ligations.</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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found. <a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/9_October_2008#fig1">Fig. 1</a>.</li>
<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/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI plasmid with NdeI and SacI (BamHI buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> with CIAP </li>
<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/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI plasmid with NdeI and SacI (BamHI buffer), <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> with CIAP </li>
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<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 - 6000 bp. <a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/9_October_2008#fig2">Fig. 2</a>. </li>
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<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: ??????. </li>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/77/Traw_petxba_omp_07_10_2008_na_9_10.jpg"></a>
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<var><b>Fig. 1.</b>XbaI/BamHI digests of pET15b+OmpA_omega<br>
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1. Marker<br>
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2-9. XbaI/BamHI digests of pET15b+OmpA_omega<br> </var>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested vector with omega_link (from 30 September) and alpha_link (from 29 September) fragments (1 hr).</li>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d3/Gelout_pET_ompa_omega.jpg"/></a>
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<var><b>Fig. 2.</b> Digests of pET15b_OmpA_omega without XbaI<br>
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1. DNA ladder<br>
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2. pET15b_OmpA_omega without XbaI digested with NdeI and SacI<br></var>
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</ol>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a></h3>
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<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#ligation">Ligation</a> of digested pET15b vector (from <A href=https://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=5&arg0=3_October_2008&arg1=6_October_2008&arg2=7_October_2008&arg3=8_October_2008&arg4=9_October_2008&name=Preparation%20of%20vector%20for%20pT7%20constructs>Preparation of vector for pT7 constructs</a>) with alpha_linker fragment(from <A href=https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008>25 September</a>) (1 hr).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on above ligation using
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PETt7L_XNE">pETt7L_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a> primers
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(annealing temperature 58 &deg;C; elongation length 120s) to obtain  <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a>fragment. </li>
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<li> Gel electrophoresis of PCR products 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 (<a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a> - 800 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/9_October_2008#fig3">Fig. 3</a>.</li>
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<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified PCR product EcoRI and SacI (BamHI buffer). </li>
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</ol>
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<p class="hide"><img src="https://static.igem.org/mediawiki/2008/2/2a/Go2_08_10_2008.jpg" width=200></a>
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<var><b>Fig. 3.</b> PCR to obtain pT7_alpha_link and pT7_omega_link<br>
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1. Marker<br>
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2. PCR to obtain alpha_linker under pT7<br>
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3. PCR to obtain omega_linker under pT7<br></var></p>
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 +
 +
 +
 +
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a></h3>
 +
<h4>Michał K.</h4>
 +
<ol>
 +
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_linker fragment(from <A href=https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008>30 September</a>) (1 hr).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on above ligations using
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on above ligations using
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#pETt7L_XNE">pETt7L_XNE</a>/<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#pETt7L_XNE">pETt7L_XNE</a>/<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a> primers separetly
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PETt7L_XNE">pETt7L_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 58 &deg;C; elongation length 120s) to obtain <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> fragments </li>
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(annealing temperature 58 &deg;C; elongation length 120s) to obtain pT7_omega_ and pT7_alpha fragments. </li>
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<li> Gel electrophoresis of PCR products 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 (pT7_omega_ - 600 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/9_October_2008#fig3">Fig. 3</a>.</li>
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<li> Gel electrophoresis of PCR products 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 (pT7_omega_ - 600 bp and alpha_link - 800 bp).</li>
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 +
<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified PCR product with EcoRI and BcuI (BamHI buffer). </li>
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</ol>
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 +
<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/2/2a/Go2_08_10_2008.jpg" width=200></a>
 +
<var><b>Fig. 3.</b> PCR to obtain pT7_alpha_link and pT7_omega_link<br>
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1. Marker<br>
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2. PCR to obtain alpha_linker under pT7<br>
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3. PCR to obtain omega_linker under pT7<br></var>
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 +
 
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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>Piotr</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 plasmid from culture inoculated on previous day <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> +<a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> (without internal EcoRI site).</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 <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> with EcoRI and PstI (Orange buffer) proper clones found.</li></ol>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a></h3>
 +
<h4>Michał K.</h4>
 +
 
 +
<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 inoculated on previous day <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</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 EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/9_October_2008#fig4">Fig. 4</a>.</li>
 +
 
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<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified PCR products: pT7_omega_ with EcoRI and BcuI (BamHI buffer) and  alpha_link with EcoRI and SacI (BamHI buffer). </li>
 
</ol>
</ol>
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<a name="fig4"><img src="https://static.igem.org/mediawiki/2008/6/66/Traw_aid_08_10_2008.jpg" width=180></a>
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<var><b>Fig.4.</b>Control EcoRI/PstI digests of pSB1A3+AID<br>
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1. Marker<br>
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2-5. Control EcoRI/PstI digests of pSB1A3+AID<br></var>
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<h3></h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a></h3>
<h4>Piotr</h4>
<h4>Piotr</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 plasmid from culture inoculated on previous day <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega (without EcoRI site).</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 <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega with EcoRI and PstI (Orange buffer) proper clones found.</li>
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<p> Inoculation of colonies from plate with ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site) to liquid LB + tetracycline.</p>
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<li> Inoculation of colonies from plate with ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> without EcoRI site to liquid LB + tetracycline.</li>
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</html>
</html>

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Preparation of vector for pT7 constructs

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day - pET15b+OmpA_omega (with removed XbaI site).
  2. Control digest of isolated pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found. Fig. 1.
  3. Digest of pET15b+OmpA_omega without XbaI plasmid with NdeI and SacI (BamHI buffer), dephosphorylation with CIAP
  4. Gel electrophoresis and gel-out of proper band - 6000 bp. Fig. 2.
  5. Fig. 1.XbaI/BamHI digests of pET15b+OmpA_omega
    1. Marker
    2-9. XbaI/BamHI digests of pET15b+OmpA_omega
    Fig. 2. Digests of pET15b_OmpA_omega without XbaI
    1. DNA ladder
    2. pET15b_OmpA_omega without XbaI digested with NdeI and SacI

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

  1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with alpha_linker fragment(from 25 September) (1 hr).
  2. PCR on above ligation using pETt7L_XNE and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 120s) to obtain alpha_linker under PT7 (BBa_K103019)fragment.
  3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker under PT7 (BBa_K103019) - 800 bp). Fig. 3.
  4. Overnight digest of purified PCR product EcoRI and SacI (BamHI buffer).

Fig. 3. PCR to obtain pT7_alpha_link and pT7_omega_link
1. Marker
2. PCR to obtain alpha_linker under pT7
3. PCR to obtain omega_linker under pT7

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_linker fragment(from 30 September) (1 hr).
  2. PCR on above ligations using pETt7L_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 120s) to obtain omega_linker under PT7 (BBa_K103020) fragments
  3. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp). Fig. 3.
  4. Overnight digest of purified PCR product with EcoRI and BcuI (BamHI buffer).
Fig. 3. PCR to obtain pT7_alpha_link and pT7_omega_link
1. Marker
2. PCR to obtain alpha_linker under pT7
3. PCR to obtain omega_linker under pT7

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Piotr

  1. Isolation of plasmid from culture inoculated on previous day pSB2K3 +BBa_K103018 (without internal EcoRI site).
  2. Control digest of isolated pSB2K3 + BBa_K103018 with EcoRI and PstI (Orange buffer) proper clones found.

Preparation of AID(BBa_K103001)

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day pSB1A3+ AID(BBa_K103001).
  2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found. Fig. 4.
Fig.4.Control EcoRI/PstI digests of pSB1A3+AID
1. Marker
2-5. Control EcoRI/PstI digests of pSB1A3+AID

Preparation of AID under pBAD/araC (BBa_K103002)

Piotr

Inoculation of colonies from plate with ligation of pMPMT5+AID (with removed EcoRI site) to liquid LB + tetracycline.