Model 3130 automated dna sequencer

Molecular cloning and determination of the nucleotide sequences of HIV-1 strains passaged in the presence of each compound were performed as previously described (15 (link)). In brief, high-molecular-weight DNA was extracted from HIV-1-infected MT-4 cells by using the InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA) and was subjected to molecular cloning, followed by sequence determination. The primers used for the PCR with the entire Gag- and protease-encoding regions of the HIV-1 genome were LTR F2 (5′-GAG ACT CTG GTA ACT AGA GAT C-3′) and Ksma2.1 (5′-CCA TCC CGG GCT TTA ATT TTA CTG GTA C-3′). The PCR mixture consisted of 1 μl of proviral DNA solution, 10 μl of Premix Taq (Ex Taq version; TaKaRa Bio Inc., Otsu, Japan), and 10 pmol of each of the PCR primers in a total volume of 20 μl. The PCR conditions used were as follows: (i) an initial step of 1 min at 95°C; (ii) 30 cycles, with 1 cycle consisting of 40 s at 95°C, 20 s at 55°C, and 2 min at 72°C; (iii) a final extension step of 10 min at 72°C. The PCR products were purified with spin columns (MicroSpin S-400 HR columns; Amersham Biosciences Corp., Piscataway, NJ), cloned directly, and subjected to sequencing with a model 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA).