Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of BioBricks

Michał K.

1. Digest of RFP(?????)+deltaA with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
2. Gel electrophoresis and gel-out of proper band 2200 bp.
3. Ligation of digested 5kb (??????) vector from (30 September) with pT7_omega_link and RFP(?????) with pT7_alpha fragment (1 hr).
4. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AlphaL+Nde and AlphaP+link10+homo2 primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha fragment.
5. Gel electrophoresis and gel-out of proper band 600 bp.

Piotr

1. Transformation of TOP10 with above ligations.
2. Tranformants planting on LB with kanamycin.
3. Isolation of plasmid from culture inoculated on previous day - pMPMT5+AID without EcoRI site.
4. Control digest of isolated plasmids with EcoRI and BamHI (BamHI buffer) - proper clones found.

Michał K.

1. Temperature gradient PCR on pMPMT5+AID without EcoRI site plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 40 - 60 °C; elongation length 2.30 min) to obtain AraC+pBAD+AID fragment. Gel electrophoresis.
2. PCR on pMPMT5+AID without EcoRI site plasmid using AraCl and AIDP_HindSpeNotPst primers (annealing temperature 45 °C; elongation length 2.30 min) to obtain AraC+pBAD+AID fragment.
3. Gel electrophoresis and gel-out of proper band 1800 bp.
4. Digest of isolated PCR product with XbaI and PstI (Tango buffer).
5. Clean-up of digested PCR product.