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coli cells were plated onto Luria-Bertani (LB) plates with 50 [micro]g/mL kanamycin and 40 [micro]g/mL each X-gal (5-bromo-4-chloro-3-indolyl-[beta]-D-galactoside or [beta]-galactosidase) and IPTG (isopropyhhio[beta]-D-galactoside) for screening of transformed cells. The amplicons were cloned with TOPO TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA), following the manufacturer's instructions, and transformed into TOP10 chemically competent Escherichia coli cells on bacterial plates containing x-gel (5-bromo-4-chloro-3-indolyl-[beta]-D-galactoside) and IPTG (isopropylthio-[beta]-D-galactoside) for blue-white screening. The plasmid was then inserted into BL21 (DE3)pLysS and expression inducted with IPTG. |
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