Team:Warsaw/Calendar-Main/25 September 2008

From 2008.igem.org

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<h3>Preparation of pT7_omega_link</h3>
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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>
<h4>Michał K.</h4>
<ol>
<ol>
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<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 <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</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 band (omega_link - 350 bp)(<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2.</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 band (omega_linker - 350 bp)(<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/25_September_2008#fig2">Fig. 2.</a>).</li>
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<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>
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<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>
<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>

Revision as of 12:18, 27 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).
  3. Measurment of concentration of both isolated products.

Preparation of ΔA (BBa_K103003)

Piotr, Michał K.

  1. Colony PCR with AL_BNXNE and APSacSpe primers on colonies from plates with transformations pSB1A3 + ΔAFig. 1. No products visible after gel electrophoresis.
  2. Inoculation of some colonies from plate to LB with ampicillin.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AlphaL+Nde and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha_linker under PT7 (BBa_K103019) 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.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmegLNde and LinP_BS primers (annealing temperature 58 °C; elongation length 45s) to obtain omega_linker under PT7 (BBa_K103020) 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. 1. Colony PCR to obtain pSB1A3 + ΔA
1. Marker
2-13. PCR on various colonies
Fig. 2. PCR to obtain
1. Marker
2. alpha_link PCR
3. omega_link PCR