Flp double cross cloning into a linear vector. a) Cloning of the lacZ cassette into linear pBA128 plasmid. The plasmid is linearized with PstI and incubated in the presence of the lacZ cassette and Flp (left). Flp catalyzes the insertion of the lacZ cassette into the linear vector forming a circular p128lacZ derivative (right). The insertion takes place in either orientation with the formation of the A and B isoforms. Colonies harboring the desired plasmids are blue when plated in the presence of X-Gal. The two isoforms can be distinguished by digestion with EcoRI. b. Agarose gel electrophoretic analysis of DNA from Flp double cross cloning of lacZ cassette into linear vectors. The lacZ cassette was incubated with linear pBA128 DNA (lanes 2–8). The DNA resulting from transformation of competent XL1-Blue cells was digested with XbaI. The DNAs in corresponding lanes shown in panels b) and c) were isolated from the same colony. M-1Kb+ marker DNAs. Blue colonies (B) all showed a 512 bp band diagnostic of insertion of the lacZ cassette (lanes 4–8) whereas the white colony (W) did not (lane 3). The parent plasmid (P, pBA128, lane2) showed band of 672 bp. c) EcoRI digestion. The isolates in lanes 5, 6, 8, showed the 512 bp band indicative of the 'B' orientation whereas those in lanes 4, and, 7 showed no such band since the 118 bp, indicative of the 'A' orientation band was not visible in this gel. Note however that the top vector band is migrating more slowly than those DNAs having the 'B' orientation. P-parental DNA was pBA128 (lane2) digested with EcoRI.