Team:Warsaw/Calendar-Main/13 October 2008

From 2008.igem.org

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<h3>Preparation of pT7_omega_link</h3>
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<h4>Michał K., Piotr</h4>
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<li> Inoculation of colonies from plate with ligation of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> + pT7_omega_link to liquid LB + kanamycin.</li></ol>
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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>
<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>
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<li> Inoculation of colonies from plate with ligation of 5kb (??????) + pT7_omega_link to liquid LB + kanamycin and RFP(?????) +  pT7_alpha fragment to liquid LB + ampicillin.</li>
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Revision as of 09:57, 27 October 2008

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Preparation of pSB2K3 + linker_alpha (BBa_K103009)

Michał K.

  1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlphaPSpe primers (annealing temperature 58 °C; elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment.
  2. Gel electrophoresis and gel-out of proper band 600 bp - linker_alpha (BBa_K103009) PCR product.
  3. Digest of isolated PCR product linker_alpha (BBa_K103009) with EcoRI and BcuI (BamHI buffer).
  4. Clean-up of digested PCR product.
  5. Overnight ligation of DNA fragments: pSB2K3 + linker_alpha (BBa_K103009).

Preparation of pACYC177 + OmpA-linker-omega-linker (BBa_K103016)

Michał K.

Inoculation of few more colonies from old plate with ligation of pACYC177 + OmpA-linker-omega-linker (BBa_K103016) to liquid LB + kanamycin.

Preparation of pT7_alpha_link

Michał K.

Inoculation of colonies from plate with ligation of pSB1A3 carrying pT7_alpha fragment to liquid LB + ampicillin.

Preparation of pT7_omega_link

Michał K., Piotr

  1. Inoculation of colonies from plate with ligation of pSB2K3 + pT7_omega_link to liquid LB + kanamycin.

Preparation of BioBricks

Michał K.

  1. Digest of 3kb(????)+pLac_OmpA_omega with BcuI and SacI (SacI buffer), dephosphorylation with CIAP.
  2. PCR on alpha PCR product from 10 September using AlphaL+SacI and LinP_BS2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha2 fragment with proper restriction sites.
  3. Gel electrophoresis and gel-out of proper band 600 bp for alpha PCR products and 3000 bp for 3kb(????)+pLac_OmpA_.
  4. Digest of isolated PCR products: alpha2 fragment with SacI and BcuI (SacI buffer) and link_alpha with EcoRI and BcuI (BamHI buffer).
  5. Clean-up of digested PCR products.
  6. Overnight ligation of DNA fragments: 3kb(?????)+ link_alpha; 3kb(????)_pLac_OmpA_ + alpha2; RFP(?????) (from 6 October) + AraC+pBAD+AID.