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 (Fig. 1) 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 test

Michał L., Ewa

1. Colony PCR.
   Template: DNA isolated from white colonies
   Primers: pZCseqL and pZCseqR
   
   

   Colony PCR program
   Temperature	Time	No. of cycles
   94°C	4:00
   94°C	0:30	28 cycles
   48°C	0:45
   72°C	1:30
   72°C	10:00
   4°C	infinite

   
   
2. DNA gel electrophoresis of PCR products. Fig. 2.
3. Gel-out of proper products (~1200 bp).
4. Sequencing of proper fragments using primer pZCseqL.

Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA

Piotr, Antoni

1. Digest of pET15b-OmpA-alpha and Z (in Geneart vector) with NdeI and NotI (Orange buffer).
2. Gel-out of Z (~200 bp band).
3. Overnight ligation of Z into digested pET15b-OmpA-alpha.