Cloning of protein Z DNA to OmpA constructs

Michał K.

1. 4 colonies from transformation pACYC177+OmpA_Z_alpha were inoculated to liquid LB broth with kanamycin

Preparation of alfa+A conctruct
Antoni

1. Gradient PCR on alpha+A
2. PCR with gradient of DMSO on alpha+A
3. gel electrophoresis

<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcl">Polymerase Chain Ligation</a> on linker-A and omega-linker

Michał L., Ewa, Marcin

• reisolated PCR product omega-linker - 4 µl
  
• reisolated PCR product linker-A - 13.5 µl
  
• primer <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI_N">OmegaL+SacI_N</a> - 2 µl
• primer <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl
• Pfu buffer with Mg²⁺ - 5 µl
• dNTPs - 1 µl
• H₂o - 22 µl
• Program:
1. 95°C - 3'
2. 95°C - 30"
3. 55°C - 45"
4. 68°C - 1'
5. go to step 2 25 x
6. 68° - 10'
   
7. keep in 4°
• gel electrophoresis of products

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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