Droplet generator dg8 cartridge

Manufactured by Bio-Rad

Sourced in United States

The Droplet Generator DG8 Cartridge is a lab equipment product designed for use with the Droplet Generator system. It is responsible for the generation of uniform, monodisperse droplets for various applications in the field of biotechnology and scientific research.

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DG8 cartridge (48 citations) Droplet generator (43 citations) Droplet generator cartridge (10 citations) DG8TM cartridges (8 citations) Droplet cartridge (3 citations) DG8™ Cartridge for QX200 Droplet Generator (2 citations)

6 protocols using droplet generator dg8 cartridge

Viral RNA Detection via ddRT-PCR

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Viral RNA extracted for the in-house assays was also analysed by ddRT-PCR. 2 μL of each sample was added to 12.5 μL of One-Step RT-ddPCR kit for probes (Bio-Rad) with 0.9 μM of each primer and 0.125μM of probe (Table 1), 1 μL of 25mM manganese acetate and molecular-grade water to a final volume of 25 μL. To generate the droplets, 20 μL of these solutions was pipetted into a Droplet Generator DG8 Cartridge (Bio-Rad) together with 70 μL of droplet generator oil for probes and loaded in the QX100 Droplet Generator (Bio-Rad). The entire droplet emulsion volume was then loaded in a twin.tec semi-skirted 96-well PCR plate (Eppendorf) and heat sealed with pierceable foil in the PX1™ PCR Plate Sealer and placed in a C1000 Touch™ Thermo Cycler (both from Bio-Rad). Thermal cycling conditions were: 30 minutes at 60°C, 5 minutes at 95°C, followed by 45 cycles of 30 seconds at 94°C and 1 minute at 60°C, and a final step of 10 minutes at 98°C.The droplets were read in a QX100™ droplet reader (Bio-Rad), and analysed using QuantaSoft™ software version 1.7.4.

Mattiuzzo G., Ashall J., Doris K.S., MacLellan-Gibson K., Nicolson C., Wilkinson D.E., Harvey R., Almond N., Anderson R., Efstathiou S., Minor P.D, & Page M. (2015). Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays. PLoS ONE, 10(11), e0142751.

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Quantitative Digital PCR for RNA Quantification

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Total RNA isolation and reverse transcription were performed as described above for qRT-PCR. Quantification by ddPCR was carried out in 20 μl reactions containing 10 μl of QX200 ddPCR EvaGreen SuperMix, 250 nM each commercial probe, 900 nM specific commercial primers, and 1 μl of cDNA according to the manufacturer’s recommendations. A negative control contained sterile double-distilled water only. Emulsified 1 nl reaction droplets were generated using a QX100 droplet generator (Bio-Rad) and a droplet generator DG8 cartridge (Bio-Rad) containing 20 μl of reaction mixture and 70 μl of ddPCR droplet generation oil (Bio-Rad) per well. Thirty-five μl of the generated droplet emulsions was transferred to 96-well PCR plates that were then heat-sealed using foil sheets. Target DNA amplification was performed by thermal cycling of the droplet emulsions as follows: initial denaturation at 95 °C for 10 min; 40 cycles of 94 °C for 30 s and 60 °C for 1 min; and then 98 °C for 10 min. The fluorescence of each thermal cycled droplet was measured using a QX100 droplet reader (Bio-Rad). Data were analysed using QuantaSoft software (Bio-Rad) after setting a threshold using the fluorescence of negative controls.

Shang L., Yan Y., Zhan Y., Ke X., Shao Y., Liu Y., Yang H., Wang S., Dai S., Lu J., Yan N., Yang Z., Lu W., Liu Z., Chen S., Elmerich C, & Lin M. (2021). A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri. NPJ Biofilms and Microbiomes, 7, 54.

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Quantifying Arbovirus RNA via ddPCR

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Each arbovirus RNA listed in Table 2 except ZIKV was quantified by droplet digital PCR (ddPCR). The complementary DNA (cDNA) of each arbovirus RNA was synthesised from an extracted RNA stock using the SuperScript III First-Strand Synthesis System (Invitrogen) with forward primer for RVFV and reverse primers for DENV, WNV, YFV, and CHIKV, respectively (Supplementary Table 1). The primers used for ddPCR were designed using Primer3 (Supplementary Table 1). All 20-μL ddPCR mixtures contained 2× EvaGreen ddPCR Supermix (Bio-Rad, Hercules, CA, USA), 0.1 μM forward and reverse primers, and 2 μL of cDNA. Each oil compartment of the droplet generator DG8 cartridge (Bio-Rad) was filled with 70 μL of droplet generation oil for EvaGreen (Bio-Rad), and approximately 20,000 droplets were generated in each well by the QX200 Droplet Generator (Bio-Rad). The reactions were performed in a 40-μL droplet emulsion using a GeneAmp PCR System 9700 (Applied Biosystems) under the following thermal cycling conditions: 95 °C for 10 min, followed by 45 cycles of 94 °C for 30 s and 60 °C for 2 min, with a final step at 98 °C for 10 min. Controls without the template were used to monitor for signals from contamination or primer-dimer formation. The cycled droplets were read individually using the QX200 droplet reader (Bio-Rad) and analysed with QuantaSoft Droplet Reader software (Bio-Rad).

Kurosaki Y., Martins D.B., Kimura M., Catena A.D., Borba M.A., Mattos S.D., Abe H., Yoshikawa R., de Lima Filho J.L, & Yasuda J. (2017). Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification. Scientific Reports, 7, 13503.

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Absolute Quantification of Gene Expression via Digital PCR

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Digital PCR was performed using the QX100 system (Bio-Rad, Hercules, CA). cDNA was transcribed from RNA treated with RNase-free DNase I (Qiagen, Maryland, MD) and an RNase inhibitor (10U/μl) (Invitrogen, Carlsbad, CA) using iScript cDNA synthesis kit (BioRad, Hercules, CA). Reaction mixtures were prepared using 10 μl ddPCR 2x Master Mix (Bio-Rad, Hercules, CA), 1 μl 20x Primer & TaqMan Probe Mix (Applied Biosystem, Foster City, CA), 5 μl nuclease free water, and 4 μl reverse-transcribed product. 1 nL reaction droplets were generated using a QX100 Droplet generator (Bio-Rad, Hercules, CA) and a droplet generator DG8 cartridge (Bio-Rad, Hercules, CA) containing 20 μl of reaction mixture and 70 μl of droplet generation oil (Bio-Rad, Hercules, CA) per well. Thirty-five microliters of the generated droplets were transferred to 96-well PCR plates and amplified using a thermal cycler as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 1 minute, and finally 98°C for 10 minutes. The fluorescence intensity of each droplet was then measured using the QX100 droplet reader (Bio-Rad, Hercules, CA). Data was analyzed using the QuantaSoft software (Version 1.3.2.0, Bio-Rad, Hercules, CA) with thresholds based on fluorescence of negative controls.

Yan I.K., Wang X., Asmann Y.W., Haga H, & Patel T. (2016). Circulating Extracellular RNA Markers of Liver Regeneration. PLoS ONE, 11(7), e0155888.

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Quantifying NRAS Mutants via ddPCR

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Digital Droplet PCR (ddPCR) reactions (20 μL) were prepared with 2X dUTP-free ddPCR supermix (Bio-Rad Laboratories Inc., Hercules, CA), 20X NRAS mutant (FAM) and wildtype (HEX) primer/probe set, and purified genomic DNA that contained approximately 60,000 copies (~200 ng) of NRAS. The following commercially available assays were validated and utilized: NRAS^G12D (dHsaMDV2010095) and NRAS^G12S (dHsaMDV2010093). To determine the limit of detection, known mutant NRAS^G12D or NRAS^G12S plasmids were titrated with NRAS^WT plasmid. For all MOLM14 parental (N = 3) and resistant cell lines (N = 4 for FGF2-derived, N = 4 for FL-derived), genomic DNA was isolated at 0, 2, 5.5, and 8.5 months. The ddPCR reaction mixture and droplet generation oil were transferred to the Droplet Generator DG8 Cartridge (Bio-Rad Laboratories Inc.) and droplets were generated with QX200 ddPCR Droplet Generator (Bio-Rad Laboratories Inc.). Droplets were transferred to a 96-well PCR plate, heat-sealed, and placed in the C1000 Touch™Thermo Cycler (Bio-Rad Laboratories). Cycling conditions were as follows: 95°C for 10 min, 40 cycles of 94°C for 30 sec, 55°C for 1 min followed by 98°C for 10 min (ramp rate 2°C/sec). Results were analyzed and visualized by QuantaSoft™ software to determine variant allele frequency. All biological replicates were merged in downstream analysis.

Joshi S.K., Nechiporuk T., Bottomly D., Piehowski P.D., Reisz J.A., Pittsenbarger J., Kaempf A., Gosline S.J., Wang Y.T., Hansen J.R., Gritsenko M.A., Hutchinson C., Weitz K.K., Moon J., Cendali F., Fillmore T.L., Tsai C.F., Schepmoes A.A., Shi T., Arshad O.A., McDermott J.E., Babur O., Watanabe-Smith K., Demir E., D’Alessandro A., Liu T., Tognon C.E., Tyner J.W., McWeeney S.K., Rodland K.D., Druker B.J, & Traer E. (2021). The AML microenvironment catalyzes a step-wise evolution to gilteritinib resistance. Cancer cell, 39(7), 999-1014.e8.

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Droplet Digital PCR Quantification

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Droplet digital PCR probe and primer sequences are same as Taqman probes. For the ddPCR experiment, a master mix was created by adding: 10 μL of ddPCR supermix for probes (Bio-Rad, United States), 2 μL of primers-probe solution (Sangon Biotech), 7 μL of ddH₂O and 1 μL of DNA of the 50-donor pool, reaching a final reaction volume of 20 μL. Twenty microliters of this solution were added to 8 compartments of the Droplet Generator DG8 Cartridge (Bio-Rad, United States) and droplets were generated. The entire droplet emulsion volume was then loaded into a 96-well PCR plate (Bio-Rad, United States). The loaded PCR plate was sealed with heat in the P X 1 PCR Plate Sealer (Bio-Rad, United States) and placed in a GeneAmp PCR System 9700 (Applied Biosystems) with the following program: incubation for 10 min at 95°C, then 40 cycles of 94°C for 30 s, 60°C for 1 min, and 98°C for 10 min. After PCR amplification, the droplets were analyzed in a QX200 droplet reader Digital PCR System (Bio-Rad, United States), and the quantification of PCR targets was analyzed using QuantaSoft™ software version 1.7.4.0917. The results were reported as the number of copies per microliter (copies/μl).

Wang Y., Chen X., Chen Q., Chen T., Chen K., Wu Y, & Wang L. (2022). SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR. Frontiers in Genetics, 13, 794285.

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