E coli top10 or stbl2

DNA isolated from the St. Maries and Virginia strains was digested with XbaI and HindIII restriction enzymes (New England Biolabs) and separated on a 0.7% agarose gel. Gel slices corresponding to the appropriate size of the aaap locus were extracted using the QIAquick Gel Extraction kit (Qiagen). The DNA was ligated with XbaI-HindIII-digested pBluescript II KS- (Stratagene) vector. Transformed colonies, grown in E. coli TOP10 or Stbl2 (Invitrogen) cells, were screened for inserts containing the aaap locus by membrane hybridization using the Digoxigenin-labeled aaap probe. Membrane hybridization was carried out as directed (Roche Applied Science) with denaturation in 0.5 M NaOH, 1.5 M NaCl for 15 min, neutralization in 0.5 M Tris [pH 7.5], 1.5 M NaCl for 15 min, followed by 10 min in 2× SSC. Hybridization was as described for Southern blots. Cloning of the St. Maries aaap locus from DNA isolated from infected ISE6 cells was performed via Polymerase Chain Reaction (PCR) using primers 5′-CAG GCC CAA AAT CGC GTC ATC C-3′ and 5′-CCC TAG CCC TAT ATC GGT TGC GAA TA-3′. The ends were cut with the restriction enzymes HindIII and XbaI and ligated into pBluescript II KS-.