Ddpcr supermix no dutp

Manufactured by Bio-Rad

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DdPCR Supermix (no dUTP) is a reagent formulation designed for use in digital droplet PCR (ddPCR) applications. It contains the necessary components for PCR amplification, including a DNA polymerase, buffer, and dNTPs, but does not contain dUTP.

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DdPCR Supermix for Probes (No dUTP) (160 citations) DdPCR Supermix (116 citations) DdPCR Supermix for Probe (no dUTP) (5 citations) DdPCR Supermix for Probes (No dUTP) kit (3 citations) DdPCR Supermix for Probes with no dUTP (2 citations)

5 protocols using ddpcr supermix no dutp

DropPCR Analysis of Transgenic HepG2 Cells

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HepG2 cells were transfected using the electroporation method described above. Four days after the electroporation, gDNA was extracted and digested using HindIII (NEB) at 37 °C for 1 h. HepG2 cells were purchased from the Cell Culture Facility at UC Berkeley (Original source: ATCC HB-8065). In each ddPCR reaction, 50–100 ng DNA, ddPCR primers, probes, 10 μl of ddPCR Supermix (No dUTP) (Bio-Rad), and nuclease-free water were added, to generate a 20 μl volume, and mixed well. Reaction mixtures, together with 70 μl QX200 Droplet generation oil, was loaded into the appropriate wells of an 8-channel droplet generation cartridge, according to the instruction manual. The cartridge was placed in the QX200™ Droplet Generator to generate the droplets, which were then transferred to a 96-well plate and amplified by standard PCR. Cycling conditions were: 95 °C for 5 min, 40 cycles of 94 °C for 30 s, and 58 °C for 1 min, followed by 98 °C for 10 min and final hold at 4 °C. The ramp rate was 2 °C/s. After thermal cycling, plates were placed in QX200™ Droplet Reader for data acquisition.

Park H.M., Liu H., Wu J., Chong A., Mackley V., Fellmann C., Rao A., Jiang F., Chu H., Murthy N, & Lee K. (2018). Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo. Nature Communications, 9, 3313.

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Quantitative Real-Time PCR and Digital PCR Protocols

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Reactions were performed according to the manufacturer’s protocol. Briefly, a reaction mixture of 1X PrimeTime Gene Expression master mix with ROX reference dye or the ddPCR Supermix (no dUTP) (Bio-Rad, USA), 900 nM of forward and reverse primers, 250 nM of corresponding probes and cDNA from the RT reactions. Thermal cycling was performed using the 7500 Fast Real-Time PCR (Thermo Fisher Scientific, USA) system set at the maximum ramp rate. The PCR cycling conditions using the PT master mix were as follows: polymerase activation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 15 s and extension at 60 °C for 1 min. The PCR cycling conditions using the ddPCR supermix were as follows: polymerase activation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 30 s, extension at 60 °C for 1 min and enzyme deactivation at 98 °C for 10 min.

Casmil I.C., Huang C, & Blakney A.K. (2023). A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA. Scientific Reports, 13, 19050.

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Quantifying HERV-K env in Biological Samples

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The ddPCR reaction was set in 96-well plates in duplicate in an AutoDG Droplet Digital PCR System (Bio-Rad) with a set of primers and probe (FAM labeled) to detect HERV-K env (forward primer, 5′-ATTTGGTGCCAGGAACTGAG-3′; reverse primer, 5′-GCTGTCTCTTCGGAGCTGTT-3′; and probe 5′-6-FAM-AGGAGTTGCTGATGGCCTCG Iowa Black FQ-3′) (Supplemental Table 12). For the analysis of CSF samples, to confirm the extracellular origin of HML-2 DNA in CSF, a premade assay of primers and probes targeting a cellular DNA (RPP30 gene, HEX-tagged) was also included (Bio-Rad, 10031244). For the analysis of HML-2 RNA in brain tissue and cell lines, HPRT1 was used as a reference gene (HEX-tagged, Bio-Rad premade assay, 10031256). The master mix was composed of 12.5 μL ddPCR Supermix (no dUTP) (Bio-Rad), 1.25 μL of a mix of HERV-K env primers (900 nm) and probe (250 nm) (Bio-Rad), 1.25 μL of RPP30 or HPRT1 assay (Bio-Rad), 2.5 μL of cDNA, and 7.5 μL of RNAse-free water. After preparing the droplets, the PCR was performed in a T100 Thermal cycler (Bio-Rad) with the following cycling conditions: 95°C for 10 minutes, 40 cycles of 95°C for 30 seconds and 60°C for 1 minute, and 95°C for 10 minutes. The number of copies was determined in a QX200 Digital PCR reader (Bio-Rad).

Shah A.H., Rivas S.R., Doucet-O’Hare T.T., Govindarajan V., DeMarino C., Wang T., Ampie L., Zhang Y., Banasavadi-Siddegowda Y.K., Walbridge S., Maric D., Garcia-Montojo M., Suter R.K., Lee M.H., Zaghloul K.A., Steiner J., Elkahloun A.G., Chandar J., Seetharam D., Desgraves J., Li W., Johnson K., Ivan M.E., Komotar R.J., Gilbert M.R., Heiss J.D, & Nath A. (2023). Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma. The Journal of Clinical Investigation, 133(13), e167929.

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Quantification of HPV Types via Digital Droplet PCR

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Digital droplet PCR (ddPCR), an established commercial method, was used to quantify reference plasmid copies and HPV copies in clinical samples. We designed new primer pairs and TaqMan probes targeting the L1 gene of HPV 45 and HPV 68, and used published primers and probes targeting the remaining 12 HR-HPVs (Table S3).^{26 (link)–27 (link)} For each ddPCR reaction, 25 μL were prepared containing1X ddPCR Supermix (No dUTP, BioRad), 0.9 μM HR-HPV type-specific primers, 0.25 μM TaqMan probe (Sigma), and 1 μL DNA template. 20 μL of each reaction was then used to generate droplets on the Bio-Rad QX200. Emulsified droplets were subjected to PCR on a Bio-Rad thermocycler using 1 cycle at 95 °C for 2 min; 45 cycles of 95 °C for 15 sec, 55 °C for 30 sec, and 72 °C for 30 sec; and 1 cycle at 37 °C for 30 sec. Subsequently, droplets were read on the Bio-Rad QX200 droplet reader and analyzed with BioRad Quantsoft Analysis Pro 1.0.596. The QX200 reads and analyzes 15000–20000 droplets per sample.

Wang J., Staheli J.P., Wu A., Kreutz J.E., Hu Q., Wang J., Schneider T., Fujimoto B.S., Qin Y., Yen G.S., Weng B., Shibley K., Haynes H., Winer R.L., Feng Q, & Chiu D.T. (2021). Detection of 14 High-risk Human Papillomaviruses Using Digital LAMP Assays on a Self-digitization Chip. Analytical chemistry, 93(6), 3266-3272.

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Quantification of Intrahepatic HBV Replication

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To assess the level of intrahepatic replication-competent HBV DNA (repDNA, defined as cccDNA and rcDNA), genome DNA was extracted from liver tissues of infected Hu-URG mice by DNeasy Blood & Tissue Kit (Qiagen, Duesseldorf, German) according to manufacturer's directions. For HBV cccDNA quantitation, intrahepatic total DNA was treated with T5 Exonuclease (NEB, MA, USA) for 1 h at 37 °C to digest single-strand DNA and linear double-strand DNA. Primers targeting the HBV DNA gap region and a fluorescence hybridization probe were used to detect the viral cccDNA and repDNA. Species-specific primers (identify human genome but not mouse genome) targeting RNase P and a fluorescence hybridization probe were used to determine the number of human hepatocytes. The primer pairs and probe were listed in Supplementary Table S2. Reaction mix was comprised of ddPCR Supermix (no dUTP) (Bio-Rad, California, USA), primers, probe and total/digested DNA sample. Reaction droplets were generated by QX200™ Droplet Generator (Bio-Rad). Intrahepatic HBV cccDNA or repDNA was amplified using T100™ Thermal Cycler (Bio-Rad). After amplification, positive and negative droplets were quantified by a QX100™ Droplet Reader (Bio-Rad) using QuantaSoft™ analysis software version 1.7.4 (Bio-Rad). Intrahepatic HBV cccDNA and repDNA values were normalized to the number of human hepatocytes.

Li G., Yang D., Liu X., Zhang T., Liu H., Zou J., Xu Z., Chen X., Dai L., Chen H, & Lu F. (2024). Precore mutation enhances viral replication to facilitate persistent infection especially in HBeAg-negative patients. Virologica Sinica, 39(2), 319-330.

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