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 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

Antoni

1. Setup of overnight culture E. coli TOP10 (LB broth) for preparation of chemocompetent bacteria.

Cloning of protein A DNA to OmpA constructs

1. Colony PCR on colonies from plates with transformations pACYC177+OmpA_A_omega and pACYC177+OmpA_A_alpha. Primers used: AL+SacI and AP+NotI
2. Confirmed transformant colonies (found only on pACYC177+OmpA_A_alpha plate) inoculated to liquid LB with kanamycin.

Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin:

1. Isolation of plasmids from transformants
2. Digest of plasmids with SacI and NotI
3. Gel electrophoresis of products
4. 2 selected clones were send to DNA sequencing lab