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

Paweł

1. Isolation of plasmids from cultures inocluated on previous day
2. Control digest of isolated plasmids with NdeI and NotI. Two positives obtained (proper band: ~200 bp visible)
3. One of positive plasmids transformed into TOP10 and plated on LB+amp, overnight, for further isolation of pET15b+Z+omega vector

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains).

Cloning of omega_A DNA fragment to pACYC177+OmpA_alpha

1. DNA fragment of omega_A isolated from gel on 20 July digestion with SacI and NotI.
2. clean-upClean-up of digested DNA fragment.
3. Digestion (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
4. Gel electrophoresis and gel-out of 4300 bp band.
5. Ligation of DNA fragments from 2. and 4. (1 hr)
6. Transformation of E. coli TOP10 strain with ligation.
7. Transformants plating on LB + kanamycin.

Cloning of protein A DNA to OmpA constructs

1. Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_A_omega).
2. Control digest of isolated plasmids with BamHI and SacI (BamHI buffer). Proper clone founded.

1. Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_A from previous day) inoculated to liquid LB with kanamycin.