MutD5 testing
Emilia
- Isolation of plasmid from culture inoculated on previous day.
- Preparation of samples to sequencing.
Mutagenesis of protein APaweł
Treatment of mutageneses as on 23rd September.
Preparation of alpha_A construct
Antoni
- 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).
- Gel electrophoresis of PCR products and gel-out of proper band (1000 bp).
Piotr, Michał K.
- Colony PCR with AL_BNXNE and APSacSpe
primers on colonies from plates with transformations pSB1A3 + ΔA. No products visible after gel electrophoresis. Fig. 1.
- Inoculation of some colonies from plate to LB with ampicillin.
Fig. 1. Colony PCR to obtain pSB1A3 + ΔA
1. Marker
2-13. PCR on various colonies
Michał K.
- PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using
AlphaL+Nde and AlphaPlinkSac
primers (annealing temperature 58 °C; elongation length 60s) to obtain alpha_linker fragment.
- Gel electrophoresis of PCR product and gel-out of proper band (alpha_linker - 600 bp). Fig. 2.
- Digest of purified PCR product with NdeI and SacI (BamHI buffer).
- 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
Michał K.
- PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using
OmegLNde and LinP_BS
primers (annealing temperature 58 °C; elongation length 45s) to obtain omega_linker fragment.
- Gel electrophoresis of PCR products and gel-out of proper band (omega_linker - 350 bp). Fig. 2.
- Digest of purified PCR product with NdeI and SacI (BamHI buffer).
- 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
|
|