Preparation of vector for pT7 constructs

Michał K.

1. Isolation of plasmids from cultures inoculated on previous day - pET15b+OmpA_omega (with removed XbaI site).
2. Control digest of isolated pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found. Fig. 1.
3. Digest of pET15b+OmpA_omega without XbaI plasmid with NdeI and SacI (BamHI buffer), dephosphorylation with CIAP
4. Gel electrophoresis and gel-out of proper band - 6000 bp. Fig. 2.
Fig. 1.XbaI/BamHI digests of pET15b+OmpA_omega
1. Marker
2-9. XbaI/BamHI digests of pET15b+OmpA_omega
Fig. 2. Digests of pET15b_OmpA_omega without XbaI
1. DNA ladder
2. pET15b_OmpA_omega without XbaI digested with NdeI and SacI

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with alpha_linker fragment(from 25 September) (1 hr).
2. PCR on above ligation using pETt7L_XNE and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 120s) to obtain alpha_linker under PT7 (BBa_K103019)fragment.
3. Gel electrophoresis of PCR products and gel-out of proper bands (alpha_linker under PT7 (BBa_K103019) - 800 bp). Fig. 3.
4. Overnight digest of purified PCR product EcoRI and SacI (BamHI buffer).

Fig. 3. PCR to obtain pT7_alpha_link and pT7_omega_link
1. Marker
2. PCR to obtain alpha_linker under pT7
3. PCR to obtain omega_linker under pT7

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_linker fragment(from 30 September) (1 hr).
2. PCR on above ligations using pETt7L_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 120s) to obtain omega_linker under PT7 (BBa_K103020) fragments
3. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp). Fig. 3.
4. Overnight digest of purified PCR product with EcoRI and BcuI (BamHI buffer).

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Piotr

1. Isolation of plasmid from culture inoculated on previous day pSB2K3 +BBa_K103018 (without internal EcoRI site).
2. Control digest of isolated pSB2K3 + BBa_K103018 with EcoRI and PstI (Orange buffer) proper clones found.

Preparation of AID(BBa_K103001)

Michał K.

1. Isolation of plasmids from cultures inoculated on previous day pSB1A3+ AID(BBa_K103001).
2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found. Fig. 4.

Preparation of AID under pBAD/araC (BBa_K103002)

Piotr

Inoculation of colonies from plate with ligation of pMPMT5+AID (with removed EcoRI site) to liquid LB + tetracycline.