The PCR amplification conditions were 1 cycle of 95uC for 1 min; 18 cycles of 95uC for 50 s, 60uC for 50 s, 68uC for 8 min; and 1 cycle of 68uC for 7 min. The 25-ml PCR mixtures contained 63 ng of each primer, 5 ng of the plasmid template, 0.2 mM deoxynucleoside triphosphate, and 16reaction buffer. PfuUltra HF DNA polymerase (Stratagene) was used, amplicons were digested with DpnI to remove contaminating template DNA, and 2 ml was transformed into maximum-efficiency XL10-Gold ultracompetent cells (5610 9 CFU/mg DNA; Stratagene). Half of the transformation was plated onto LB-ampicillin agar plates, generally resulting in 10 to 50 colonies per reaction. All mutants were confirmed by sequencing the entire env gene.