Plasmid purification maxi prep kit

HeLa cells (ATCC) were maintained in Dulbecco modified Eagle medium supplemented with 10% (vol/vol) fetal bovine serum (FBS). The Jurkat T cell line (ATCC) was maintained in RPMI 1640 supplemented with 10% (vol/vol) FBS. All media were supplemented with 2 mM l-glutamine. Plasmids used were the infectious HIV-1 molecular clone pNL4-3 (63 (link)); two derivatives with point mutations at SP1 residue 3 (SP1-A3V and SP1-A3T) (39 (link)); four derivatives with point mutations in CA at residues 156 (CA-G165E), 157 (CA-P157S), 160 (CA-P160L), and 225 (CA-G225D) (kind gift from Eric Freed, NIH, USA) (4 (link)); and two protease-defective derivatives, pNL4-3/PR⁻ with the active-site mutation PR-D25N, which contains either wild-type (WT) Gag or the SP1-A3V mutation (64 (link)). Plasmid DNA was purified with a plasmid purification maxiprep kit (Qiagen) and adjusted to 1 μg/μl. HeLa cells were transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and Jurkat T cells were transfected by using DEAE-dextran (65 (link)).

Bacterial artificial chromosome (BAC) clones were purchased from BACPAC resources centre, C.H.O.R.I. BAC DNA was isolated using QIAGEN® Plasmid Purification MaxiPrep kit. A volume of 1 μg of BAC clone DNA was labelled by nick translation for 90 min at 15 °C with digoxigenin-11-dUTP (Roche) using a nick translation kit (Roche) or ATTO labelling kit (Jena Bioscience). Labelled probe together with cot-1 DNA (Roche) was dehydrated. The denatured probe was re-suspended in hybridisation buffer and placed on a metaphase slide (prepared as described earlier), which had been dehydrated in a graded ethanol series (70%, 85% and 100%). The slide was sealed, denatured (82.5 °C for 2 min) and incubated O/N at 37 °C. The slide was washed at 65 °C in 0.1 × SSC. DIG-labelled probe was detected with FITC-conjugated anti-digoxigenin for 30 min at RT. Chromosomes were counterstained with DAPI Vectasheild.