isogenic strain of genotype y; cn bw sp according to the methods described in Smoller et al., (1991) and Lozovskaya et al., (1993). High molecular weight DNA was partially digested with Sau3A1 and fractionated according to molecular weight in sucrose gradients (10–40% sucrose) in order to isolate fragments in the size range 75–100 kb. These fractions were dialyzed, concentrated with 2-butanol, and precipitated in ethanol in preparation for ligation to the P1 vectors.
P1 Cloning Vectors. P1 clones were produced by using the P1 vectors pNS582-tet14 Ad10 (Figure 1A) and pAd10 sacBII (Figure 1B), which differ in the region around the cloning site. In pNS582-tet14 Ad10 (Sternberg, 1990), the BamHI cloning site interrupts the tetracycline-resistance gene; in pAd10 sacBII (Pierce et al., 1992), the BamHI cloning site is flanked on one side by a T7 promoter, a Not I site, and the promoter of the sacBII gene of Bacillus amyloliquefaciens; and it is flanked on the other side by an SP6 promoter, an Sfi I site, and the sacBII structural gene for levansucrase. Large fragments of DNA inserted into the cloning site disrupt expression of the sacBII gene and thereby allow cells of E. coli to survive in medium containing 5% sucrose (Pierce et al., 1992).
Vector arms resulting from digestion of either vector with BamHI and Sca I were treated with calf intestinal alkaline phosphatase, and an equimolar ratio of vector-arm DNA and genomic DNA was ligated in the presence of T4 DNA ligase as described (Smoller et al., 1991). Packaging of the ligated DNA was carried out as described in Sternberg (1990). During packaging, molecules are packaged stepwise in the counterclockwise direction from the pac site (as Figure 1 is drawn) until the phage head has been filled (100–115 kb), after which cleavage occurs.