Visit in US Embassy

Visas have been accorded to the whole team.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K., Piotr

1. Clean-up of overnight digest reaction.
2. Digest of pSB1A3 carrying ΔA (BBa_K103003) with EcoRI and SacI (BamHI buffer), dephosphorylation with CIAP
3. Gel electrophoresis and gel-out of proper band - 2200 bp.
4. Ligation of digested pSB1A3 with alpha_linker under PT7 (BBa_K103019) fragment (1 hr).
5. Transformation of TOP10 with above ligation.
6. Tranformants plating on LB with ampicillin.

Preparation of pT7_omega_link

Michał K., Piotr

1. Ligation of digested pSB2K3 vector from (30 September) with pT7_omega_link fragment (1 hr).
2. Transformation of TOP10 with above ligation.
3. Tranformants plating on LB with kanamycin.

Preparation of BioBricks

Michał K.

1. 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.
2. Gel electrophoresis and gel-out of proper band 600 bp.

Piotr

1. Transformation of TOP10 with above ligations.
2. Tranformants plating 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.