Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

1. Isolation of pKS+omega_ΔA from culture inoculated on previous day.
2. Digest of pKS+omega_ΔA with SacI and NotI (BamHI buffer) (Fig.1.).
3. Digest (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
4. Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_ΔA) bands.
5. Ligation of DNA fragments from 2. and 3. (1 hr)
6. Transformation of E. coli TOP10 strain with ligation.
7. Transformants plating on LB + kanamycin.

Purification of proteins: Z-alpha and Z-omega

Piotr

pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain inoculated in liquid LB with chloramphenicol and ampicillin.