Dna engine peltier thermal cycler

Genotyping was conducted on SNPs in the genes ANK3 and CACNA1C (Supplementary Table 1). Allele-specific primers and Sanger sequencing primers were designed for each SNP using BatchPrimer3 (Supplementary Table 2). PCR amplification was performed using 10× PCR buffer (Invitrogen, Catalog # 10342020), 1.5nM of MgCl₂, 0.2mM dNTPs, 0.5μM primer, 1 unit of Taq DNA polymerase (Invitrogen, Catalog # 10342020) and 20ng genomic DNA. Sequence amplification was performed with a Bio-Rad DNA Engine Peltier Thermal Cycler with the following cycling conditions: initial denaturation at 95°C for 5mins followed by 30 cycles of 95°C for 30sec, 58°C for 30sec, and 72°C for 45sec, with a final elongation of 72°C for 5min. The PCR products were run on a 1.5% agarose gel, and randomly selected samples were sequenced to confirm the specificity of allele specific primers using allele flanking primers (Supplementary Table 2, Figure 1). For sequencing, bands were cut from the gel and DNA was extracted using the QIAGEN MinElute gel extraction kit (Cat. No. 28604). Samples underwent Sanger sequencing at the Keck DNA Sequencing Lab at Yale University, and electropherograms were visualized using 4Peaks (Figure 1).

Two-step nested PCR assays were used for quantitative HIV-1 DNA integration analysis as previously described (Friedrich et al., 2010 (link); Iordanskiy et al., 2010 (link)). The first round was performed in a 25 μl reaction mix to pre-amplify the genomic DNA from infected macrophages. The following primers were used: Alu forward, 5’-GCC TCC CAA AGT GCT GGG ATT ACA G-3’; and HIV-1 gag reverse, 5’-GCT CTC GCA CCC ATC TCT CTC C-3’. The PCR reaction contained 10x EX Taq Buffer, dNTP mix (2.5 mM), EX Taq (all from TaKaRa Bio Inc., Otsu, Shiga, Japan), ddH₂O, 100 nM Alu forward primer, 600 nM gag reverse primer, and 100 ng of genomic DNA. The DNA Engine Peltier Thermal Cycler (Bio Rad, Hercules, CA) was programmed to perform a 2-minute hot start at 94°C, followed by 30 steps of denaturation at 93°C for 30 second, annealing at 50°C for 1 minute, and extension at 70°C for two minutes. The second round real-time quantitative PCR was performed by using 5.6 μl of the reaction mix from the pre-amplification step. The amplified DNA was standardized with an internal GAPDH control. The sequences of the primers and probe were as follows: LTR forward, 5’-GCC TCA ATA AAG CTT GCC TTG A-3’; LTR reverse, 5’ -TCC ACA CTG ACT AAA AGG GTC TGA-3’; probe: 5’-FAM-GCG AGT GCC CGT CTG TTG TGT GAC TCT GGT AAC TAG CTC GC-BHQ-3’.