A MOTHER vector carrying a gene encoding KringleYFP was mixed with an E. coli expression DAUGHTER vector in the presence of Electra Reagent Mix for 5–60 minutes. Reactions were transformed into E. coli and plated onto LB plates supplemented with appropriate selective antibiotics (Figure 1).
Transformants resulted from reactions that were incubated for as little as 5 minutes and maximum efficiency was obtained by 40 minutes (Table 1). Almost all transformants showed an inducible fluorescent yellow phenotype, indicating accurate movement of the gene from the MOTHER into the expression DAUGHTER vectors. DAUGHTER constructs are selected because of antibiotic-resistance markers.
MOTHER vectors carry a counter-selection marker, either rpsL (streptomycin sensitivity)1 or PheS (phenylalanine analog p-chlorophenylalanine sensitivity)2 that allows selection against carryover of MOTHER vector by plating transformants onto media that contains both DAUGHTER selection antibiotic and MOTHER counter-selection agent.
In a separate experiment, crude PCR product of the KringleYFP gene was successfully cloned into an E. coli DAUGHTER vector without any PCR reaction treatment or cleanup, using a single-tube 5 minute protocol.