a). Cloning of a 1.65 Kb fragment of human DNA into receptor plasmid p128lacZ. Schematic diagram. A 1.65 Kb fragment from intron 10 of the human KCNQ1 gene (dashed arc) was amplified using the primers 3 and 4 and subjected to Flp double cross cloning into receptor plasmid p128lacZ. The resultant recombinants transform XL1-Blue cells to form white colonies (right). The KCNQ1 fragment replaces the lacZ cassette in either orientation. In the 'A' orientation EcoRI digestion yields a 118 bp fragment while in the 'B' orientation a 1.65 Kb fragment is seen. b) Agarose gel analysis of DNA isolated from white colonies after Flp double cross cloning of KCNQ1 fragment into 128lacZ receptor plasmid. XbaI. Six of the plasmids from 8 white colonies (lanes 2–4, 6, 7 and 9) show the expected 1.6 Kb fragment. Lane 1-plasmid pBA128; lane 10-marker DNAs (M, 1 Kb+marker DNA). c) The DNAs shown in b were then digested with EcoRI (1anes 1–9). Lanes 2–7 show the same DNAs as in panel b) from lanes 2, 3, 4, 6, 7, 9. Four of them show a 1.5 Kb fragment diagnostic of the B form of the plasmid (lanes 2, 4, 5, 7,) whereas the DNA from two of the six is in the A form (lanes 3 and 6,). The 118 bp fragment cannot be seen on this gel. Lane 9-1Kb+ marker DNAs.