Preparation of BioBricks

Michał K.

1. Isolation of plasmids from culture inoculated on previous day (pSB1A3+Z(BBa_K103004)).
2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found.
3. Digest of pLac_OmpA_omega fragment with EcoRI and BcuI (BamHI buffer).
4. Gel elctrophoresis and gel-out of proper band - 1200 bp.
5. Colony PCR with OmpaL_N and OmpaP_link primers on colonies from plates with transformations pSB1A3+OmpA (annealing temperature - 55°C,45 s of elongation step).
6. Gel electrophoresis.
7. Confirmed transformant colonies inoculated to liquid LB with ampicillin.
8. Overnight ligation of isolated DNA fragments: pSB2K3 + _alpha, pSB2K3 + _omega and pACYC177 + OmpA_omega_ (both digested with BamHI and PstI - from 30 September).