Team:Warsaw/Calendar-Main/6 October 2008

From 2008.igem.org

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<h3>Preparation of pLac_OmpA_omega</h3>
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<h4>Michał K.</h4>
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<h3>Preparation of BioBricks</h3>
<h3>Preparation of BioBricks</h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<p> 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/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega (without EcoRI site).</p>
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<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/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega (without EcoRI site) and self ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI.</li>
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Revision as of 10:37, 27 October 2008

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Preparation of linker_alpha (BBa_K103009)

Michał K.

  1. Colony PCR with LinLSXNE and AlphaPSpe primers on colonies from plates with transformations pSB1A3+ linker_alpha (BBa_K103009) (annealing temperature - 55°C,60 s of elongation step). No visible bands for proper product.
  2. Inoculation of some colonies which grown on plate - pSB2K3 + linker_alpha (BBa_K103009) to liquid LB + kanamycin.

Preparation of linker_omega (BBa_K103013)

Michał K.

Inoculation of some colonies which grown on plate - pSB2K3 + linker_omega (BBa_K103013) to liquid LB + kanamycin.

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

Michał K.

  1. Colony PCR with OmpaL_N and OmpaP_link primers on colonies from plates with transformations pACYC177 + OmpA-linker-omega-linker (BBa_K103016) (annealing temperature - 55°C,90 s of elongation step). No visible bands for proper product.
  2. Inoculation of some colonies which grown on plate transformation: pACYC177 + OmpA-linker-omega-linker (BBa_K103016) to liquid LB + kanamycin.

Preparation of vector for pT7 constructs

Michał K.

Overnight selfligation of pET15b+OmpA_omega without XbaI.

Preparation of pLac_OmpA_omega

Michał K.

Preparation of BioBricks

Michał K.

Overnight ligation of isolated DNA fragments pSB2K3 + pLac_OmpA_omega (without EcoRI site).

  • Digest of pSB1A3 plasmid with XbaI and PstI (Tango buffer). Dephosphorylation (CIAP) of plasmid.
  • Gel electrophoresis and gel-out of proper band - 2200 bp.