Preparation of constructs with OmpA protein fusions

Michał K.

1. PCR on pB30D plasmid with OmpaL_N and OmpaP_link primers (15 cycles, elongation duration 45 s, annealing temperature 63°C).
2. PCR on pUC19 plasmid with AlphaL_link and AlphaP_XB primers (20 cycles, elongation duration 45 s, annealing temperature 63°C).
3. PCR on pUC19 plasmid with OmegaL_link and OmegaP_EPB primers (20 cycles, elongation duration 30 s, annealing temperature 58°C).
   As a result we got three PCR products: OmpA_linker and two fragments of TEM1 beta-lactamese: linker_alpha and linker_omega.
4. Gel electrophoresis of PCR products and gel-out of proper bands (OmpA_linker - 500 bp, linker_alpha - 600 bp and linker_omega - 350 bp).
5. Electrophoresis to estimate the concentration of isolated DNA.

Blue/white and rifampicin test

Michał L., Ewa, Marcin:

1. Colony PCR
   Template: DNA isolated from white colonies
   Primers: pZCseqL and pZCseqR
   Annealing time:
   Number of cycles:
2. DNA gel electrophoresis of PCR products.
3. Gel-out of proper products (~1200 bp).
4. Sequencing of proper fragments using primer pZCseqL.