Cloning of protein Z DNA to OmpA constructs
Michał K.

1. Digest and dephosphorylation (with CIAP) of pACYC177+OmpA_alpha with SacI and NotI.
2. Gel eloctrophoresis and gel-out (4300 bp band).
3. Ligation of isolated DNA fragment and Z DNA fragment isolated on 10 July.
4. Transformation of E. coli TOP10 strain with ligation.
5. Transformants plating on LB + kanamycin.

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 of protein Z DNA to OmpA constructs

2 colonies was inoculated to liquid LB broth with kanamycin

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

1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega).
2. Control digest of isolated plasmids with BamHI and SacI (we found good clones for both ligations).

Polymerase Chain Ligation 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 OmegaL+SacI_N - 2 µl
• primer AP+NotI_N - 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