Team:Warsaw/Calendar-Main/6 October 2008

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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/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> (annealing temperature - 55&deg;C,60 s of elongation step). No visible bands for proper product.</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/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> (annealing temperature - 55&deg;C,60 s of elongation step). No visible bands for proper product.</li>
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<li>Inoculation of some colonies which grown on plate - <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _alpha to liquid LB + kanamycin. </li></ol>
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<li>Inoculation of some colonies which grown on plate - <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> to liquid LB + kanamycin. </li></ol>

Revision as of 23:24, 26 October 2008

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Preparation of pSB2K3 + _alpha

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 pSB2K3 + _omega

Michał K.

Inoculation of some colonies which grown on plate - pSB2K3 + _omega to liquid LB + kanamycin.

Preparation of BioBricks

Michał K.

  1. Colony PCR with OmpaL_N and OmpaP_link primers on colonies from plates with transformations pSB1A3+ OmpA_omega (annealing temperature - 55°C,90 s of elongation step). No visible bands for proper product.
  2. Inoculation of some colonies which grown on plates with three separate transformations: pSB2K3 + _alpha, pSB2K3 + _omega and pACYC177 + OmpA_omega_ to liquid LB + kanamycin.
  3. Digest of pSB1A3 plasmid with XbaI and PstI (Tango buffer). Dephosphorylation (CIAP) of plasmid.
  4. Gel electrophoresis and gel-out of proper band - 2200 bp.
  5. Overnight ligation of isolated DNA fragments: pSB2K3 + pLac_OmpA_omega (without EcoRI site) and self ligation of pET15b+OmpA_omega without XbaI.