Team:Warsaw/Calendar-Main/25 September 2008

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<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>
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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>
  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>
<li>Inoculation of some colonies from plate to LB with ampicillin.</li></ol>
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<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>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a></h3>
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103019>alpha_linker under PT7 (BBa_K103019)</a></h3>

Revision as of 16:53, 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 + 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_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. 2. PCR to obtain
1. Marker
2. alpha_link PCR
3. omega_link PCR