Preparation of vector for pT7 constructs

Michał K.

1. Isolation of plasmids from cultures inoculated on previous day - pET15b+OmpA_omega without XbaI.
2. Control digest of isolated pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found.
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.

Preparation of pT7_alpha_link

Michał K.

1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with alpha_link 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 pT7_alpha fragment.
3. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_alpha_link - 800 bp).
4. Overnight digest of purified PCR product EcoRI and SacI (BamHI buffer).

Preparation of pT7_omega_link

Michał K.

1. Ligation of digested pET15b vector (from Preparation of vector for pT7 constructs) with omega_link fragment(from 30 September) (1 hr).
2. Ligation of digested vector with omega_link (from 30 September) and alpha_link (from 29 September) fragments (1 hr).
3. PCR on above ligations using pETt7L_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 120s) to obtain pT7_omega_ fragments
4. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp).
5. Overnight digest of purified PCR product with EcoRI and BcuI (BamHI buffer).

Preparation of BioBricks

Michał K.

1. Isolation of plasmids from cultures inoculated on previous day pSB1A3+ AID and pACYC177 + pET15b+OmpA_omega without XbaI).
2. Control digest of isolated pSB1A3+ AID plasmids with EcoRI and PstI (Orange buffer) and pET15b+OmpA_omega without XbaI plasmids with XbaI and BamHI (Tango buffer). Gel electrophoresis - proper clones found for both ligations.

Piotr

1. Isolation of plasmid from culture inoculated on previous day pSB2K3 + pLac_OmpA_omega (without EcoRI site).
2. Control digest of isolated pSB2K3 + pLac_OmpA_omega with EcoRI and PstI (Orange buffer) proper clones found.
3. Inoculation of colonies from plate with ligation of pMPMT5+AID without EcoRI site to liquid LB + tetracycline.