Team:Warsaw/Calendar-Main/25 September 2008

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<h4>Emilia</h4>
<h4>Emilia</h4>
<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.</li>
<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.</li>
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<li>preparation of samples to sequencing</li>
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<li>Preparation of samples to sequencing.</li>
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 +
</ol>
<h3>Mutagenesis of protein A</h3><h4>Paweł</h4>
<h3>Mutagenesis of protein A</h3><h4>Paweł</h4>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha_linker and linker_A with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers and 10% DMSO (30 cycles, elongation 60&nbsp;s, annealing temperature 72&deg;C). </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha_linker and linker_A with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> primers and 10% DMSO (30 cycles, elongation 60&nbsp;s, annealing temperature 72&deg;C). </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 (1000 bp).</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 (1000 bp).</li>
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<li>Measurment of concentration of both isolated products.</li>
 
</ol></p>
</ol></p>
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<h3>Preparation of BioBricks</h3>
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<h4>Michał K.</h4>
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 +
 
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<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a></h3>
 +
<h4>Piotr, Michał K.</h4>
<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#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>
<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#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>
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  primers on colonies from plates with transformations RFP(??????????) + deltaA. No products visible after gel electrophoresis.</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/wiki/index.php?title=Part:BBa_K103003>&Delta;A</a>. No products visible after gel electrophoresis. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig1">Fig. 1</a>.</li>
 +
<li>Inoculation of some colonies from plate to LB with ampicillin.</li></ol>
 +
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/b/bf/Kolonijny_25_09.jpg" width=300/></a><var><b>Fig. 1.</b> Colony PCR to obtain pSB1A3 + ΔA<br>
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1. Marker<br>
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2-13. PCR on various colonies<br></var>
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</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
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+Nde">AlphaL+Nde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a>
 +
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha_linker fragment. </li>
 +
<li> Gel electrophoresis of PCR product 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 (alpha_linker - 600 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2</a>.</li>
 +
 +
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified PCR product with NdeI and SacI (BamHI buffer). </li>
 +
 +
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product.</li></ol>
 +
 +
<p class="hide"><br>
 +
<img src="https://static.igem.org/mediawiki/2008/8/8b/Go_25_09.jpg" width=300/><var><b>Fig. 2. Results of PCR to obtain alpha_linker and omega_linker</b>:<br>
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1. Marker<br>
 +
2. alpha_link PCR <br>
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3. omega_link PCR<br></var>
 +
</p>
 +
 +
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a></h3>
 +
<h4>Michał K.</h4>
 +
<ol>
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<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  
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<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#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>
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  primers (annealing temperature 58 &deg;C; elongation length 45s) to obtain omega_link fragment. </li>
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  primers (annealing temperature 58 &deg;C; elongation length 45s) to obtain omega_linker 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
+
-
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+Nde">AlphaL+Nde</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaPlinkSac">AlphaPlinkSac</a>
 
-
primers (annealing temperature 58 &deg;C; elongation length 60s) to obtain alpha_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/25_September_2008#fig2">Fig. 2</a>.</li>
 +
 +
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#Digest">Digest</a> of purified PCR product with NdeI and SacI (BamHI buffer). </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 bands (omega_link - 350 bp and alpha_link - 600 bp).</li>
+
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR product.</li></ol>
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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: omega_link with EcoRI and BcuI (BamHI buffer) and  alpha_link with NdeI and SacI (BamHI buffer). </li>
 
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digested PCR products.</li></ol>
 
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<h3></h3>
 
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<h4>Piotr</h4>
 
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<p>Inoculation of some colonies from plate to LB with ampicillin.</p>
 
 +
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/8/8b/Go_25_09.jpg" width=300/></a><var><b>Fig. 2.</b> Results of PCR to obtain alpha_linker and omega_linker:<br>
 +
1. Marker<br>
 +
2. alpha_link PCR <br>
 +
3. omega_link PCR<br></var>

Latest revision as of 03:48, 29 October 2008

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MutD5 testing

Emilia

  1. Isolation of plasmid from culture inoculated on previous day.
  2. Preparation of samples to sequencing.

Mutagenesis of protein A

Paweł

Treatment of mutageneses as on 23rd September.

Preparation of alpha_A construct

Antoni

  1. PCR on alpha_linker and linker_A with AlphaL+SacI and AP+NotI primers and 10% DMSO (30 cycles, elongation 60 s, annealing temperature 72°C).
  2. Gel electrophoresis of PCR products and gel-out of proper band (1000 bp).

Preparation of ΔA (BBa_K103003)

Piotr, Michał K.

  1. Colony PCR with AL_BNXNE and APSacSpe primers on colonies from plates with transformations pSB1A3 + ΔA. No products visible after gel electrophoresis. Fig. 1.
  2. Inoculation of some colonies from plate to LB with ampicillin.
Fig. 1. Colony PCR to obtain pSB1A3 + ΔA
1. Marker
2-13. PCR on various colonies

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AlphaL+Nde and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha_linker fragment.
  2. Gel electrophoresis of PCR product and gel-out of proper band (alpha_linker - 600 bp). Fig. 2.
  3. Digest of purified PCR product with NdeI and SacI (BamHI buffer).
  4. Clean-up of digested PCR product.


Fig. 2. Results of PCR to obtain alpha_linker and omega_linker:
1. Marker
2. alpha_link PCR
3. omega_link PCR

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmegLNde and LinP_BS primers (annealing temperature 58 °C; elongation length 45s) to obtain omega_linker fragment.
  2. Gel electrophoresis of PCR products and gel-out of proper band (omega_linker - 350 bp). Fig. 2.
  3. Digest of purified PCR product with NdeI and SacI (BamHI buffer).
  4. Clean-up of digested PCR product.
Fig. 2. Results of PCR to obtain alpha_linker and omega_linker:
1. Marker
2. alpha_link PCR
3. omega_link PCR