Mutagenesis of protein A

Paweł

Treatment of mutageneses as on 23rd September.

Preparation of alpha_A construct

Antoni

1. PCR on alpha_linker and linker_A with AlphaL+SacI and AP+NotI primers and 10% DMSO (30 cycles, elongation 60 s, annealing temperature 72°C).
2. Gel electrophoresis of PCR products and gel-out of proper band (1000 bp).
3. Measurment of concentration of both isolated products.

Preparation of BioBricks

Michał K.

1. Colony PCR with AL_BNXNE and APSacSpe primers on colonies from plates with transformations RFP(??????????) + deltaA. No products visible after gel electrophoresis.
2. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmegLNde and LinP_BS primers (annealing temperature 58 °C; elongation length 45s) to obtain omega_link fragment.
3. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AlphaL+Nde and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha_link fragment.
4. Gel electrophoresis of PCR products and gel-out of proper bands (omega_link - 350 bp and alpha_link - 600 bp).
5. Overnight digest of purified PCR products: omega_link with EcoRI and BcuI (BamHI buffer) and alpha_link with NdeI and SacI (BamHI buffer).
6. Clean-up of digested PCR products.

Piotr

Inoculation of some colonies from plate to LB with ampicillin.