Preparation of linker_alpha (BBa_K103009)

Michał K.

1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlphaPSpe primers (annealing temperature 58 °C; elongation length 60s) to obtain linker_alpha (BBa_K103009) fragment.
2. Gel electrophoresis and gel-out of proper band 600 bp - linker_alpha (BBa_K103009) PCR product.
3. Digest of isolated PCR product linker_alpha (BBa_K103009) with EcoRI and BcuI (BamHI buffer).
4. Clean-up of digested PCR product.
5. Overnight ligation of DNA fragments: pSB2K3 + linker_alpha (BBa_K103009).

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

Inoculation of few more colonies from old plate with ligation of pACYC177 + OmpA-linker-omega-linker (BBa_K103016) to liquid LB + kanamycin.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

Inoculation of colonies from plate with ligation of pSB1A3 carrying alpha_linker under PT7 (BBa_K103019) fragment to liquid LB + ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

Inoculation of colonies from plate with ligation of pSB2K3 + omega_linker under PT7 (BBa_K103020) to liquid LB + kanamycin.

Preparation of AID under pBAD/araC (BBa_K103002)

Michał K.

Overnight ligation of DNA fragments: pSB1A3 (from 6 October) + AID under pBAD/araC (BBa_K103002) (from 10 October).

Preparation of OmpA_linker_alpha_linker under Plac (BBa_K103017)

Michał K.

1. Digest of pSB2K3 + OmpA_linker_omega_linker under Plac with BcuI and SacI (SacI buffer), dephosphorylation with CIAP.
2. PCR on alpha PCR product from 10 October using AlphaL+SacI and LinP_BS2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha2 fragment with proper restriction sites (SacI and BcuI).
3. Gel electrophoresis and gel-out of proper bands (600 bp for alpha2 PCR product and 3000 bp for pSB2K3 + OmpA_linker under PLac promoter fragment).
4. Digest of isolated PCR product (alpha2 fragment) with SacI and BcuI (SacI buffer).
5. Clean-up of digested PCR product.
6. Overnight ligation of DNA fragments: pSB2K3 with lactose promoter and OmpA_linker + alpha2.