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204 Caldwell et al.will allow the selection of cells carrying first one and then the other resistancemarker (see Notes 3 and 4).For cloning, the purified DNA and pKS are digested with the appropriate REovernight using the standardized insert/vector RE reaction mixture. Vectors arefurther alkaline phosphatase treated to block religation. The digested fragmentsare then isolated through agarose gel electrophoresis. The product is cut out ofthe gel and purified by the gel purification method of one’s choice. It is foundthat ligation efficiency is increased and background colonies decreased when allfragments are gel isolated for cloning. Alternatively, the target arm fragmentsmay be cloned straight from the PCR product using Invitrogen’s TOPO TACloning kit before subcloning into the pKS targeting vector (see Note 5).3.1.4. Ligation and TransformationFor ligation, the Takara DNA Ligation Kit is used as noted in Subheading 2.For transformation, use 50 µL of chemically competent DH5α cells. This isfound to be quite robust, but the method used at this point is not critical.3.1.5. Colony ScreeningTo screen colonies, one can either use plasmid preparation followed by REanalysis or perform colony PCR using a primer specific for the insert and one forthe vector. RE analysis should still be performed on PCR-identified clones forunambiguous verification. Colony PCR is performed as defined in Wahl et al. (6).1. Pick bacterial colonies and suspend in 50 µL sterile H 2O. After using for colonyPCR, this can be stored at 4°C until selected colonies are inoculated into bacterialculture media for further use. Storage time is up to 2 wk without supplementingwith bacterial culture media.2. PCR amplify using the amplification reaction mixture.PCR program:95°C 10 min95°C 30s ⎫⎪43°C 30s ⎬ 5 cycles72°C 3min⎪⎭93°C 30s ⎫⎪53°C 30 s ⎬30 cycles72°C 3min⎪⎭72°C 7 min4°C ∞