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

Antoni:

1. Preparation of chemocompetent bacteria E. coli TOP10.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

Sequencing confirms that we have obtained proper construct with omega-A fusion

Cloning of protein A DNA to OmpA constructs

1. Colony PCR on colonies from plates with transformations pACYC177+OmpA_A_omega. Primers used: AL+SacI and AP+NotI
2. Confirmed transformant colonies inoculated to liquid LB with kanamycin.
3. Isolation of plasmids from cultures inocluated on previous day (pACYC177+OmpA_A_alpha).
4. Control digest of isolated plasmids with BamHI and SacI.

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

1. Ligations transformed into TOP10 and plated on LB + ampicillin.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

1. We have obtained white colonies of transformants and inoculated 10 of them on liquid LB+Amp₁₀₀

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: ~150 bp visible)
3. One of positive plasmids transformed into TOP10 and plated on LB+amp, overnight, for further isolation of pET15b+Z+omega vector