Cloning of protein Z DNA to OmpA constructs
Michał K.

1. Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_Z_alpha).
2. Control digest of isolated plasmids with BamHI and SacI (we found 4 good clones).

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.

Cloning of protein A DNA to OmpA constructs

1. Digest of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)
2. Gel electrophoresis and gel-out of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane).
3. Ligation of A DNA fragment with both pACYC177 vectors.
4. Transformation of E. coli TOP10 strain with ligations.
5. Transformants plating on LB + kanamycin.

Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA
Paweł

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