Preparation of vectors for Biobricks

Michał K., Piotr

1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
3. Gel elctrophoresis and gel-out of proper bands: 4500 bp - pSB2K3 and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector).

Preparation of pSB1A3+Z(BBa_K103004)

Michał K.

Inoculation of some colonies from pSB1A3+Z(BBa_K103004) plate to LB with ampicillin.

Preparation of BioBricks

Michał K.

1. Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of pLac_OmpA_omega fragment. Ligation for 1 hr.
2. PCR on pMPMT5+AID plasmid using AIDl and AIDP_HindSpeNotPst primers (annealing temperature 58 °C; elongation length 60s) to obtain AID fragment.
3. PCR on ligation of pLac_OmpA_omega fragment using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain pLac_OmpA_omega fragment without EcoRI.
4. Gel electrophoresis of PCR products and gel-out of proper bands (AID - 600 bp and pLac_Omp_omega_ - 1200 bp).