Tests for ampicillin resistance given by protein added to medium

Piotr

Inoculation of TOP10 carrying pACYC177+OmpA_A_alpha, pACYC177+OmpA_ΔA_alpha, pACYC177+OmpA_ΔA_omega, pACYC177+OmpA_A_omega, pACYC177+OmpA_omega_ΔA, pACYC177+OmpA_alpha, pACYC177+OmpA_omega and pACYC177+OmpA_omega_ΔA_alpha to LB with inductor (0.25 mM IPTG) and kanamycin.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

1. Clean-up of overnight digest reaction.
2. Ligation of digested vectors from 14 August and alpha DNA fragment.
3. Electroporation of E. coli TOP10 with ligations products.
4. Transformants plating on LB + kanamycine.

Plasmid preparation for cloning of protein A DNA to pET15b+OmpA-alpha

Antoni

1. Isolation of plasmid from cultures inocluated on previous day (pET15b+OmpA-alpha).
2. Digest of pET15b+OmpA_alpha with NdeI and NotI (Orange buffer) and dephosphorylation with CIAP.
3. Gel electrophoresis (Fig. 1) and gel-out of 6300 bp band.

Cloning of protein A DNA to GeneArt plasmid

Antoni

1. Isolation of plasmids from bacterial cultures inoculated on previous day (pKS+A and pGeneart+Z).
2. Digest of pKS+A and pGeneart+Z with SacI and NotI (BamHI buffer), pGeneart dephosphorylation with CIAP.
3. Gel electrophoresis (Fig. 1) of digested plasmids and gel-out of proper bands (470 bp for protein A lane and 2570 for pGeneart)
4. Overnight ligation of pGeneart and A.

Fig. 1.Digest product - pGeneart+Z (lanes 1-3), pKS+A (4-6), pET15b+OmpA-A (8-10). Lane 7 - GeneRuler™ DNA Ladder Mix