Team:Warsaw/Calendar-Main/7 October 2008

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Preparation of pSB2K3 + _alpha

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pSB2K3 + _alpha).
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.
  3. Inoculation of few more colonies which grown on plates with three separate transformations: pSB2K3 + _alpha to liquid LB + kanamycin.

Preparation of BioBricks

Michał K.

  1. Isolation of plasmid from culture inoculated on previous day (pSB2K3 + _alpha, pSB2K3 + _omega and pACYC177 + OmpA_omega_).
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.
  3. Overnight ligation of DNA fragments: pSB1A3+ AID.

Piotr

  1. Transformation of TOP10 with ligations pSB2K3 + pLac_OmpA_omega (without EcoRI site) and pET15b+OmpA_omega without XbaI.
  2. Plating: pSB2K3 + pLac_OmpA_omega (without EcoRI site) on LB with kanamycin and pET15b+OmpA_omega without XbaI on LB with ampicillin.
  3. Inoculation of few more colonies which grown on plates with three separate transformations: pSB2K3 + _alpha, pSB2K3 + _omega and pACYC177 + OmpA_omega_ to liquid LB + kanamycin.
  4. Digest of pMPMT5+AID with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.
  5. Overnight ligation of pMPMT5+AID without EcoRI site.