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 + _alpha 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.