Preparation of alfa+A conctruct
Antoni

1. Protein A PCR on pKS+A
2. PCR on alpha
3. Gel electrophoresis
4. Gel-out
5. PCR on alpha+A

Cloning of protein Z DNA to OmpA constructs

1. Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).
2. Gel electrophoresis and gel-out of proper bands 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane).
3. Ligation of pACYC177+OmpA_omega and Z (1 hr).
4. Transformation of E. coli TOP10 strain with ligation.
5. Transformants plating on LB + kanamycin.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

We had to start form scratch with this one.

1. PCR A in 50 µl
   template DNA - pKS-A4 1 µl
   primer AP+NotI_N - 2 µl
   primer AL+link10+homo2_N - 2 µl
   Pfu polymerase buffer + Mg²⁺ (from Fermentas) - 5 µl
   dNTPs - 1 µl
   Pfu turbo - 0.5 µl
   H2o - 38.5 µl
   
   Program:
   
   1. 95°C 3'
   2. 95°C 30"
   3. 62°C 45"
   4. 72°C 45"
   5. 72°C 10'
   6. keeping in 4°C
2. PCR omega in 50 µl
   template DNA - pUC19 1 µl
   primer OmegaLS - 2 µl
   primer AOmegaPli - 2 µl
   Pfu polymerase buffer + Mg²⁺ (from Fermentas) - 5 µl
   dNTPs - 1 µl
   Pfu turbo - 0.5 µl
   H2o - 38.5 µl
   Program:
   
   1. 95°C 3'
   2. 95°C 30"
   3. 62°C 45"
   4. 72°C 45"
   5. 72°C 10'
   6. keeping in 4°C
   25 cycles
3. Gel electrophoresis
4. Reisolation from agarose gel

Preparation of constructs with OmpA protein fusions and Cloning of protein Z DNA to OmpA constructs

1. <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega).
2. Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and SacI (we found good clones for both ligations).

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