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 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.
3. Ligation of digested vector with omega_link (from 30 September) and alpha_link (from 29 September) fragments (1 hr).
4. PCR on above ligations using pETt7L_XNE/LinP_BS and pETt7L_XNE/AlphaPlinkSac primers separetly (annealing temperature 58 °C; elongation length 120s) to obtain pT7_omega_ and pT7_alpha fragments.
5. Gel electrophoresis of PCR products and gel-out of proper bands (pT7_omega_ - 600 bp and alpha_link - 800 bp).
6. Overnight digest of purified PCR products: pT7_omega_ with EcoRI and BcuI (BamHI buffer) and alpha_link with EcoRI and SacI (BamHI buffer).

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.