Allprep dna rna procedure

Genomic DNA (gDNA) and total RNA were isolated from patient PBMCs using the AllPrep DNA/RNA procedure (Qiagen). gDNA was isolated from HEK293T and TZM-bl cell lines using the QIAamp DNA mini procedure (Qiagen) 48 hr after transfection as previously described by the manufacturer. 125 ng of patient, HEK293T, or TZM-bl gDNA was utilized for a two-round, nested PCR using the high-fidelity Phusion polymerase for amplifying the HIV-1 LTRs. LTR primers for round one (LTR 3, 4) and round two (LTR 5, 6) were designed to optimally amplify patient-specific LTRs. Round one cycling conditions were as follows; 98 °C for 3 min, then 25 cycles at 98 °C for 10 sec, 50 °C for 20 sec, 72 °C for 18 sec, followed by a 10 min extension at 72 °C and a 4 °C hold. Either 2.5 or 10 μl from round one was used for the second round of PCR. Round two cycling conditions were; 98 °C for 3 min, then 30 cycles of 98 °C for 10 sec, 50 °C for 20 sec, 72 °C for 18 sec, followed by a 10 min extension at 72 °C and a 4 °C hold. Production of LTR products was confirmed by 1–2% agarose gel electrophoresis. Amplimers were sent for Sanger-based sequence confirmation using either LTR primers 5, 6 or 7 by GeneWiz. PCR products were either sent out uncleaned or following clean-up using either gel extracted PCR products, column cleanup or ExoZap. Sequences for LTR primers are provided in Table S1.

At each 6-month visit, blood was collected: One gray-top tube was sent for drugs-of-abuse screening (~10 mL) and four purple-top BD vacutainer tubes containing K2-EDTA as the anticoagulant (Becton Dickinson & Co., Franklin Lakes, NJ) were used to collect blood from patients (~40 mL) for serum and PBMC isolation, as described [41 (link), 42 (link)]. From 5×10⁶ PBMCs, genomic DNA and total RNA isolation was performed using a Qiagen (Venlo, Limburg, Netherlands) AllPrep DNA/RNA procedure as described by the manufacturer.