pQE 30-UA

Protocol tips

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	For expression, the recombinant clones were grown in LB broth containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml) with constant shaking. Cultures were induced by 1 mM isopropyl thiogalactoside (IPTG) and grown until the OD600 reached 0.5–0.7.	Whole cell protein (WCP) sample was prepared and analyzed by running 12% SDS-PAGE to check the recombinant expression of OmpA protein.

Protocol tips
For expression, the recombinant clones were grown in LB broth containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml) with constant shaking. Cultures were induced by 1 mM isopropyl thiogalactoside (IPTG) and grown until the OD600 reached 0.5–0.7.
Downstream tips
Whole cell protein (WCP) sample was prepared and analyzed by running 12% SDS-PAGE to check the recombinant expression of OmpA protein.

Publication protocol

For cloning, the portion of ompA gene excluding the region coding for the signal peptide was amplified using ompA-F2 and ompA-R primers. The PCR conditions were same as described in earlier section except that the annealing temperature was 51 °C. Purified PCR product was ligated with commercially available pQE 30-UA linearized vector (Qiagen) at 16 °C for 2 h. After ligation, plasmid was transformed into chemically competent M15 E. coli followed by heat shock and grown in LB agar with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Colonies harboring recombinant plasmid were identified and further confirmed by PCR using gene and/or vector specific primer. For expression, the recombinant clones were grown in LB broth containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml) with constant shaking. Cultures were induced by 1 mM isopropyl thiogalactoside (IPTG) and grown until the OD600 reached 0.5–0.7. Whole cell protein (WCP) sample was prepared and analyzed by running 12% SDS-PAGE to check the recombinant expression of OmpA protein. Non-recombinant M15 E. coli cells and uninduced recombinant clone were used as negative controls.

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