The presence of bacterial 16S rRNA or fungal 28S rRNA was detected separately using the QX200 Droplet Digital PCR system (Bio‐Rad, Hercules, CA, USA) according to the manufacturer’s protocol. The ddPCR mastermix had a final volume of 22 µl and contained 1× ddPCR Supermix for Residual DNA Quantification (Bio‐Rad), 900 nM of forward and reverse primer, 250 nM probe, 2 µl (approximately 50 ng µl−1) of isolated DNA and DNase‐free water. In total, 20 µl of the total mixture was used to generate droplets using the QX200 Droplet Generator (Bio‐Rad). Subsequently, the droplet suspension was transferred into a 96‐wells plate and PCR amplification was performed on a C1000 Terminal Cycler (Bio‐Rad) with the following cycling parameters: 95°C for 10 min, 40 cycles (with ramp rate 2°C s−1) of 95°C for 30 s and 61°C for 1 min, and a final step at 98°C for 10 min. The droplets were analysed on the QX200 Droplet Reader (Bio‐Rad), and results were visualized with QuantaSoft software version 1.7.4 (Bio‐Rad). Thresholds were set manually above the negative (signal‐arm) droplets. Droplets above the threshold were determined positive (signal‐rich). A sample was defined positive when a higher number of droplets/concentration (copies µl−1) was found, when compared to background control samples. The latter included pooled whole blood samples of healthy volunteers or water.
Ddpcr supermix for residual dna quantification
Manufactured by Bio-Rad
Sourced in Spain
The DdPCR Supermix for Residual DNA Quantification is a reagent designed for the detection and quantification of residual DNA in biological samples using droplet digital PCR (ddPCR) technology. The supermix contains all the necessary components for the ddPCR reaction, including DNA polymerase, buffer, and dNTPs.
Automatically generated - may contain errors
Lab products found in correlation
Qx100 droplet reader, by Bio-Rad (2 mentions)
Qx200 droplet reader, by Bio-Rad (1 mentions)
Qx200 droplet generator, by Bio-Rad (1 mentions)
Quantasoft software version 1, by Bio-Rad (1 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (1 mentions)
Quantasoft v 1, by Bio-Rad (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Ddpcr supermix for probe, by Bio-Rad (1 mentions)
3 protocols using ddpcr supermix for residual dna quantification
1
Droplet Digital PCR for Microbial RNA Detection
2
Quantifying HIV-1 DNA in Purified Lymphoid Cells
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Lysed extracts from purified LPLs (CD45+ cells), bulk LPs and PBMCs from the same participants were simultaneously obtained and used to measure cell-associated HIV-1 DNA by ddPCR. A total of 2–3 μl of cell lysates were mixed with ddPCR supermix and primers/probes (FAM/HEX-ZEN-Iowa BlackFQ double-quenched probes, Integrated DNA Technologies) in a total volume of 20 μl to generate droplets, according to manufacturer’s recommendations. We managed to circumvent potential primer mismatch in individuals’ viral sequence by using two different primers/probe sets annealing to the 5’LTR and GAG conserved regions of HIV-1 genome [21 (link)–23 (link)] (Table 1 ). Here, ddPCR Supermix for Residual DNA quantification was used (186–4037, Bio-Rad). Annealing/extension step was set at 57°C to quantify vDNA using the C1000 Touch™ Thermal Cycler (Bio-Rad) and subsequently analyzed using a QX100™ droplet reader (Bio-Rad) and the QuantaSoft v.1.6 software (Bio-Rad).
PBMCs, bulk LPs and LPLs samples were quantified in duplicate or triplicate, respectively. PBMCs from HIV-negative donors were used as negative controls and assayed in each plate to set the positive/negative threshold for ddPCR analysis, and the number of those negative control wells was the same than replicas for each sample. The RPP30 cellular gene was quantified in parallel to normalize sample input (Table 1 ) [24 (link)].
PBMCs, bulk LPs and LPLs samples were quantified in duplicate or triplicate, respectively. PBMCs from HIV-negative donors were used as negative controls and assayed in each plate to set the positive/negative threshold for ddPCR analysis, and the number of those negative control wells was the same than replicas for each sample. The RPP30 cellular gene was quantified in parallel to normalize sample input (
3
Quantifying Intact HIV Proviruses
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