Team:Warsaw/Calendar-Main/29 September 2008

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<h3>MutD<sub>5</sub> testing</h3>
<h3>MutD<sub>5</sub> testing</h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
-
<p>Sequencing results: there weren't any mutations in plasmids isolated from MutD<sub>5</sub> - maybe this strain is too weak a mutator.</p>
+
<p>Sequencing results: there weren't any mutations in plasmids isolated from MutD<sub>5</sub> - maybe this strain is too weak as a mutator.</p>
-
<h3>Preparation of BioBricks</h3>
+
<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>
-
<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of Z-GeneArt and RFP(????)+deltaA plasmids with NdeI and BcuI (BamHI buffer).</li>
 
-
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of RFP(????)+deltaA plasmid with NdeI and SacI (BamHI buffer).</li>
 
-
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmids.</li>
+
<ol>
-
<li> Gel electrophoresis of digested plasmids 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 (Z - 200 bp and both RFP (??????)'s - 2200 bp).</li>
+
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using
-
<li> Gel electrophoresis to estimate concentration of purified DNA from previous day. </li>
+
-
<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments: RFP(??????) + Z and RFP(????) + OmpA_link (from 18 September). </li>
+
-
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmLNXNEB">OmLNXNEB</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinPBSNP">LinPBSNP</a>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmLNXNEB">OmLNXNEB</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinPBSNP">LinPBSNP</a>
-
primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain OmpA_omega fragment. </li>
+
primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> fragment. </li>
-
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
+
<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 (OmpA-linker-omega-linker - 900 bp).
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_XNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
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<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig1">Fig. 1</a>. </li></li>
-
primers (annealing temperature 58 &deg;C; elongation length 90s) to obtain pLac_OmpA_omega fragment. </li>
+
</ol>
-
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on pACYC177+OmpA_omega_deltaA_alpha plasmid using
+
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>
+
<p class="hide"><img src="https://static.igem.org/mediawiki/2008/e/ef/Go_29_09_2008.jpg"width=150/>
 +
<var><b>Fig. 1. </b> PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.</var></p>
-
primers (annealing temperature 58 &deg;C; elongation length 45s) to obtain deltaA fragment. </li>
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a></h3>
 +
<h4>Michał K.</h4>
 +
 +
<ol>
 +
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a>  plasmid with NdeI and SacI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li>
 +
 +
 +
 +
<li> Gel electrophoresis of digested plasmids 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 band (<A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig2">Fig. 2</a>. </li>
 +
 +
<li> 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/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103006>OmpA_linker (BBa_K103006)</a> (from 18 September). </li></ol>
 +
 +
 +
<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#pcr">PCR</a> on <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegLNde">OmegLNde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a>
+
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegLNde">OmegLNde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (annealing temperature 55 &deg;C; elongation length 60s) to obtain omega_linker fragment. </li>
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primers (annealing temperature 55 &deg;C; elongation length 60s) to obtain omega_link fragment. </li>
+
<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 band (omega_linker - 350 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig1">Fig. 1</a>.</li></ol>
-
<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 (deltaA - 250 bp, pLac_Omp_omega_ - 1200 bp, omega_linker - 350 bp and Omp_omega_ - 900 bp).</li>
+
 
 +
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/ef/Go_29_09_2008.jpg"width=180/></a>
 +
<var><b>Fig. 1. </b> PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.</var>
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a></h3>
 +
<h4>Michał K.</h4>
 +
<ol>
 +
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneArt-Z</a> and <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a> plasmids with NdeI and BcuI (BamHI buffer). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid. </li>
 +
 
 +
 
 +
 
 +
<li> Gel electrophoresis of digested plasmids  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 (Z - 200 bp and  <A href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> - 2200 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig2">Fig. 2</a> and <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig3">Fig. 3</a>.</li>
 +
<li> 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/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></ol>
 +
 
 +
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d1/Go2_30_09_2008na29_09.jpg" width=160/></a><var><b>Fig. 2.</b> Digests of plasmid pSB1A3<br>
 +
1. Marker<br>
 +
2. pSB1A3 digested NdeI/SacI <br>
 +
3. pSB1A3 digested NdeI/BcuI <br></var>
 +
<a name="fig3"><img src="https://static.igem.org/mediawiki/2008/e/e5/Go_Z_25_09_2008.jpg" width=150/></a><var><b>Fig. 3.</b> Digest of plasmid pGeneArt-Z<br>
 +
1. Marker<br>
 +
2. pGeneArt-Z digested NdeI/BcuI <br></var>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a></h3>
 +
<h4>Michał K.</h4>
 +
<ol>
 +
 
 +
 
 +
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using
 +
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#PlacL_XNE">PlacL_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 90s) to obtain <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> fragment. </li>
 +
 
 +
 
 +
 
 +
 
 +
<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 band (BBa_K103018 - 1200 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_September_2008#fig4">Fig. 4</a>.</li>
</ol>
</ol>
-
<h3></h3>
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<a name="fig4"><img src="https://static.igem.org/mediawiki/2008/1/19/Go22_29_09.jpg" width=180/></a><var><b>Fig. 4.</b>Result of PCR on pLac_OmpA_omega_<br>
 +
1. Marker<br>
 +
2. PCR product<br></var>
 +
 
 +
 
 +
<h3>Preparation of vectors for Biobricks</h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
-
<p>Inoculation of bacteria received from iGEM HQs (         ).</p>  
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<p>Inoculation of bacteria received from iGEM HQs, carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a> plasmids.</p>  
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
</html>
</html>

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MutD5 testing

Piotr

Sequencing results: there weren't any mutations in plasmids isolated from MutD5 - maybe this strain is too weak as a mutator.

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

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmLNXNEB and LinPBSNP primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA-linker-omega-linker (BBa_K103016) fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper bands (OmpA-linker-omega-linker - 900 bp). Fig. 1.

Fig. 1. PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.

Preparation of OmpA-linker (BBa_K103006)

Michał K.

  1. Digest of pSB1A3 carrying ΔA (BBa_K103003) plasmid with NdeI and SacI (BamHI buffer). Dephosphorylation (CIAP) of plasmid.
  2. Gel electrophoresis of digested plasmids and gel-out of proper band (pSB1A3 - 2200 bp). Fig. 2.
  3. Overnight ligation of isolated DNA fragments: pSB1A3 + OmpA_linker (BBa_K103006) (from 18 September).

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. PCR on pACYC177 + OmpA_omega plasmid using OmegLNde and LinP_BS primers (annealing temperature 55 °C; elongation length 60s) to obtain omega_linker fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper band (omega_linker - 350 bp). Fig. 1.
Fig. 1. PCR products: lane 2 - Omega_Link, lane 3 - OmpA_Omega_Link.

Preparation of Z(BBa_K103004)

Michał K.

  1. Digest of pGeneArt-Z and pSB1A3 carrying ΔA (BBa_K103003) plasmids with NdeI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmid.
  2. Gel electrophoresis of digested plasmids and gel-out of proper bands (Z - 200 bp and pSB1A3 - 2200 bp). Fig. 2 and Fig. 3.
  3. Overnight ligation of isolated DNA fragments: pSB1A3 + Z (BBa_K103004).
Fig. 2. Digests of plasmid pSB1A3
1. Marker
2. pSB1A3 digested NdeI/SacI
3. pSB1A3 digested NdeI/BcuI
Fig. 3. Digest of plasmid pGeneArt-Z
1. Marker
2. pGeneArt-Z digested NdeI/BcuI

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_linker_omega_linker under Plac (BBa_K103018) fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper band (BBa_K103018 - 1200 bp). Fig. 4.
Fig. 4.Result of PCR on pLac_OmpA_omega_
1. Marker
2. PCR product

Preparation of vectors for Biobricks

Piotr

Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.