Whole blood dna extraction kit

For each patient, 500 μl of blood was collected in an EDTA microtainer tube. After 5 min of centrifugation, 200 μl of plasma was separated from the blood cell pellet and replaced by an equivalent amount of 1X PBS, pH 7.4. Using the whole blood DNA extraction kit from Qiagen, DNA was isolated from the PBS resuspended blood cell pellet and total DNA concentration was measured by NanoDrop (Thermo Fisher Scientific). We used digital droplet PCR (ddPCR) to determine EBV load in each sample by amplifying EBV BALF5 and human β-actin gene, using primers and probes shown in Table 1. The duplex ddPCR reactions were prepared in a total volume of 20 μl which contained 10 μl of ddPCR Supermix for probes (No UTP) (Bio-Rad Laboratories), and 2 sets of each primer and probe combination (0.9 μM of primers and 0.25 μM of probes). The BioRad Automated Droplet Generator (AutoDG) (Bio-Rad Laboratories) was used to ensure consistent droplet generation. After the ddPCR reaction, the number of positive and negative droplets were counted by the Bio-Rad QX200TM Droplet reader and EBV viral loads quantified as copies/ng human DNA.

Jurkat T cells were transfected with pNL4-3 WT and mutant derivatives. Compounds (5 μM) were added at the time of transfection and were maintained throughout the course of the experiment. The transfected Jurkat T cells were split every Monday, Wednesday, and Friday; supernatants were collected at each time point; and viral replication was monitored by RT activity as previously described (66 (link)). Cell pellets were harvested on the days of peak RT activity, and genomic DNA was extracted by using a whole-blood DNA extraction kit (Qiagen). Maintenance of SP1 or CA mutations and acquisition of additional mutations were investigated by amplification of the entire Gag-PR coding region using forward and reverse primers NL516F (5′-TGC CCG TCT GTT GTG TGA CTC-3′) and NL2897R (5′-AAA ATA TGC ATC GCC CAC AT-3′), respectively. The resultant 2.3-kb PCR product was purified by using a QIAquick PCR purification kit (Qiagen) and then sequenced by using primers NL1410F (5′-GGA AGC TGC AGA ATG GGA TA-3′) and NL1754F (5′-TGG TCC AAA ATG CGA ACC-3′).