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). Fig. 1.

Preparation of Z(BBa_K103004)

Michał K.

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

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

1. Addition of 5 μl T4 ligase buffer and 2 μl of T4 ligase to digest of BBa_K103018 fragment. Ligation for 1 hr.
2. PCR on ligation of OmpA_linker_omega_linker under Plac (BBa_K103018) fragment using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain BBa_K103018 fragment without internal EcoRI site (Przanowski' method of removing internal restriction site).
3. Gel electrophoresis of PCR product and gel-out of proper bands (BBa_K103018 - 1200 bp). Fig. 2.
4. Digest of PCR product with XbaI and PstI (Tango buffer).
5. Clean-up of above digest reaction.

Fig. 2. PCR to obtain AID and OmpA_linker_omega_linker under Plac
1. Marker
2. AID
3. OmpA_linker_omega_linker under Plac

Preparation of AID(BBa_K103001)

Michał K.

1. PCR on pMPMT5+AID plasmid using AIDl and AIDP_SpeNotPst primers (annealing temperature 58 °C; elongation length 60s) to obtain AID(BBa_K103001) fragment.
2. Gel electrophoresis of PCR product and gel-out of proper bands (AID(BBa_K103001) - 600 bp). Fig. 2.
3. Digest of PCR product with XbaI and PstI (Tango buffer).
4. Clean-up of above digest reaction.