MutD₅ testing

Emilia

1. Isolation of plasmid from culture inoculated on previous day.
2. preparation of samples to sequencing

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 ΔA (BBa_K103003)

Piotr, Michał K.

1. Colony PCR with AL_BNXNE and APSacSpe primers on colonies from plates with transformations pSB1A3 + ΔA. No products visible after gel electrophoresis. Fig. 1.
2. Inoculation of some colonies from plate to LB with ampicillin.

Preparation of alpha_linker under PT7 (BBa_K103019)

Michał K.

1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AlphaL+Nde and AlphaPlinkSac primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha_linker under PT7 (BBa_K103019) fragment.
2. Gel electrophoresis of PCR product and gel-out of proper band (alpha_linker - 600 bp). Fig. 2.
3. Digest of purified PCR product with NdeI and SacI (BamHI buffer).
4. Clean-up of digested PCR product.

Fig. 2. Results of PCR to obtain alpha_linker and omega_linker:
1. Marker
2. alpha_link PCR
3. omega_link PCR

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmegLNde and LinP_BS primers (annealing temperature 58 °C; elongation length 45s) to obtain omega_linker fragment.
2. Gel electrophoresis of PCR products and gel-out of proper band (omega_linker - 350 bp). Fig. 2.
3. Digest of purified PCR product with NdeI and SacI (BamHI buffer).
4. Clean-up of digested PCR product.