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.