Abi prism bigdye terminator cycle

HIV-1 RNA was isolated from plasma using the Qiagen QIAamp viral RNA kit and reverse transcribed to cDNA using SuperScript III reverse transcriptase (Invitrogen, CA). The envelope genes were amplified from single-genome templates (68 (link)), and amplicons were directly sequenced using the ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) and resolved on an ABI 3100 automated genetic analyzer. The full-length env sequences were assembled and edited using Sequencher version 4.5 software (Genecodes, Ann Arbor, MI). Multiple sequence alignments were performed using Muscle (69 (link)) and edited with AliView (70 (link)).

The bacterial genome was extracted using a DNA nucleic acid extraction kit (Tiangen, China). The 16S rRNA gene was amplified using the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’)[17 (link)]with the Q5 Hot Start High-Fidelity 2×Master Mix(NEB, U.K.). After gel electrophoresis, specific PCR products were purified using a pEASY purification kit (Transgene, China). Connection and transformation were performed using the pEASY^TM-Blunt Simple Cloning Kit (Transgene, China). The transformed bacteria were grown at 37°C for 12h on Luria-Bertani agar, containing X-gal (20 mg/ml), IPTG (24 mg/ml), and kanamycin (50 mg/ml). Five white single colonies were randomly selected for sequencing. All were sequenced with an ABI Prism BigDye Terminator cycle sequencing ready reaction kit using AmpliTaq DNA polymerase and an ABI Prism 377xl DNA sequencer (Applied Biosystems, Foster City, CA, USA) according to the instructions of the manufacturer at Tsingke BioTech Co., Ltd., sequencing the amplicons in both directions.