Topo ta cloning vector pcr2

293T cells were cotransfected with HIV-1 expression vectors and A3G expression vectors as indicated. Culture supernatants were collected 48 h post-transfection and treated with DNase I (20 U/ml) at 37°C for 1 h. SupT1 cells (1 × 10⁶) were spin-infected with DNase I-treated HIV-1 (150 ng p24^Gag equivalent) at 2000 × g for 2 h at room temperature. The cells were then washed with fresh medium and cultured at 37°C, 5% CO₂ atmosphere. Cells were harvested 12 h post-infection. DNA was isolated using a DNeasy Blood and Tissue DNA isolation kit (QIAGEN). A 650-bp DNA fragment covering a portion of nef, U3, and R of HIV-1 was amplified with Taq DNA polymerase (Invitrogen) using the primers HIV-1-F (5′-AGGCAGCTGTAGATATTAGCCAC) and HIV-1-R (5′-GTATGAGGGATCTCTAGCTACCA). The PCR products were cloned into the TOPO TA-cloning vector pCR2.1 (Invitrogen). The clones were sequenced, and the sequencing results were analyzed using the CLC Main Workbench software. Statistical analysis was performed using the GraphPad Prism 5 software.

Total RNA was isolated from 5 × 10⁶ hybridoma cells using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using the SuperScript III first-strand synthesis system for reverse transcription PCR (Invitrogen) by following the manufacturer’s instructions. PCR was performed with GoTaq hot start green master mix (Promega) using the cDNA as the template. The sense primers for the VH and VL regions anneal to the leader sequence, whereas the VH and VL antisense primers anneal to the IgG1 heavy-chain and immunoglobulin kappa light-chain constant regions, respectively (Table 1). The PCR products were purified with Wizard SV gel and PCR clean-up system (Promega) and cloned into the TOPO TA cloning vector pCR2.1 (Invitrogen) by following the manufacturer’s instructions. After cloning, plasmids were purified using the Wizard plus SV minipreps DNA purification system (Promega) and sequenced by Eurofins Scientific (Germany). The VH and VL of SagA.01 were sequenced by GenScript. The resulting sequences were analyzed using NCBI IgBLAST (https://www.ncbi.nlm.nih.gov/igblast).