A bacteriophage P1 library containing 75-100kb genomic DNA fragments. Generated to provide useful molecular genetic reagents and an intermediate level of resolution for physical mapping of the Drosophila genome.
DNA was isolated from nuclei prepared from adult fly tissue. DNA was purified by CsCl gradient ultracentrifugation. High molecular weight DNA was then partially digested with Sau3A under conditions that produced 70-150kb fragments. DNA was purified using sucrose gradient, then separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis and DNA in the size range of 75-100kb was isolated, dialyzed and ethanol precipitated. DNA fragments were then ligated into the phage P1 cloning vector pNS582-tet14Ad10 (cleaved by BamHI and ScaI). Ligated DNA was then packaged into phage heads in vitro and used to infect Cre-expressing E. coli (strain NS3145). Host cell Cre catalyzes recombination at LoxP sites in the linear recombinant molecules to generate a circular molecule. Recombinant clones are recognized by their kanamycin-resistant and tetracyline-sensitive phenotypes.
Approximately 1.7% of the clones contain sequences that hybridize with ribosomal DNA.
P1 clone sequencing was stopped when BAC clones were determined to be better substrates for sequencing. P1 clones were generated in 1991, and BAC clones were generated in 1998; over these years, some differences in transposable element insertion sites were identified.
