Figure 5.

Insertion of fragment ‘A-Ins’ into AD169-a-Del. (A) Schematic view of the insertion (paste) strategy. Fragment ‘A-Ins’ was released by digestion with SmiI+MssI. Then it was purified and transformed into competent E. coli strain GS1783 carrying AD169-a-Del3 BAC or AD169-a-Del4 BAC. Clones were selected with kanamycin and chloramphenicol. The kan^R/I-SceI cassette was removed by the second step of en passant mutagenesis. (B) BAC DNA was analyzed by restriction digestion with HindIII and EcoRI. Arrows indicate the expected alterations in the ‘A’ inserted genome (clones 3-2, 4-1 and 4-2) compared to the parental AD169 (AD) BAC. M, molecular weight standard.