Ddpcr supermix for probe no dutp

Manufactured by Bio-Rad

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The DdPCR Supermix for Probe (no dUTP) is a pre-formulated reaction mix designed for droplet digital PCR (ddPCR) applications. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and a buffer system optimized for probe-based detection in a droplet-based format, without the inclusion of dUTP.

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DdPCR Supermix for Probes (No dUTP) (160 citations) DdPCR Supermix (116 citations) Supermix for probes (15 citations) DdPCR Supermix (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 for probe no dutp

Droplet Digital PCR for EML4-ALK Detection

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cDNA was synthesized in the same methods described in the Quantitative RT-PCR section.
cDNA and EML4-ALK specific probes ddPCR EXD Assay EML4-ALK (Bio-Rad Laboratories, dHsaEXD86850342, Hercules, CA, USA) were mixed with ddPCR Supermix for Probe (no dUTP) (Bio-Rad Laboratories). Droplet was generated from the cDNA and probe mixture using Droplet Generator (Bio-Rad Laboratories) according to the manufacturer’s instructions. PCR was performed using C1000 Touch thermal cycler (Bio-Rad Laboratories) with the cycle described in the manufacturer’s protocol of the probes. After PCR, measurement of droplet was performed using Droplet Reader (Bio-Rad Laboratories) and QuantaSoft Analysis Pro Software (Bio-Rad Laboratories) was used for analysis.

Shimizu Y., Okada K., Adachi J., Abe Y., Narumi R., Uchibori K., Yanagitani N., Koike S., Takagi S., Nishio M., Fujita N, & Katayama R. (2022). GSK3 inhibition circumvents and overcomes acquired lorlatinib resistance in ALK-rearranged non-small-cell lung cancer. NPJ Precision Oncology, 6, 16.

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Digital Droplet PCR for SARS-CoV-2 Detection

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QX200 droplet generator (Bio-Rad, #1864002) was used to make emulsions following the manufacture’s instruction. Briefly, 20 μL reaction mix was prepared using ddPCR Supermix for Probe (no dUTP) (Bio-Rad, #1863024), N2 outer primers (F: AAC ACA AGC TTT CGG CAG AC, R:CCC GAA GGT GTG ACT TCC AT; final concentration of 500 nM) and template (2019-nCoV_N_Positive Control, Integrated DNA Technologies, #10006625). The ddPCR reaction mix was added to the droplet generator and converted to droplets with the use of Droplet Generation Oil for Probes (Bio-Rad, #1863005) and DG8 Cartridges and Gaskets (Bio-Rad, # 1864007).

Sun C., Liu L., Vasudevan H.N., Chang K.C, & Abate A.R. (2021). Accurate bulk quantitation of droplet digital PCR. bioRxiv.

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Droplet Digital PCR for SARS-CoV-2 Detection

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ddPCRs were performed on the QX200 Droplet Digital PCR system according to the manufacturer's instructions (Bio‐Rad). Briefly, reaction mixture consisted in 10 μl ddPCR Supermix for probe no dUTP (1863023, Bio‐Rad), 0.25‐1 ng of cDNA, primers and probes for E/IP4 and N/nsp13 duplex reactions used at concentration listed in Appendix Table S2 in a final volume of 20 μl. PCR amplification was conducted in a iCycler PCR instrument (Bio‐Rad) with the following condition: 95°C for 10 min, 40 cycles of 94°C for 30 s with a ramping of 2°/s, 59°C for 1 min with a ramping of 2°/s, followed by 98°C for 5 min with a ramping of 2°/s and a hold at 4°C. After amplification, the 96‐well plate was loaded onto the QX200 droplet reader (Bio‐Rad) that measures automatically the fluorescence intensity in individual droplets. Generated data were subsequently analyzed with QuantaSoft™ software (Bio‐Rad) based on positive and negative droplet populations. Data are expressed as CPD (copy per droplets) normalized to γ‐actin and Hprt reference gene relative expression.

de Melo G.D., Lazarini F., Larrous F., Feige L., Kornobis E., Levallois S., Marchio A., Kergoat L., Hardy D., Cokelaer T., Pineau P., Lecuit M., Lledo P., Changeux J, & Bourhy H. (2021). Attenuation of clinical and immunological outcomes during SARS‐CoV‐2 infection by ivermectin. EMBO Molecular Medicine, 13(8), e14122.

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Validating Differentially Expressed Genes by ddPCR

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The validation of a subset of genes resulting differentially expressed from the transcriptome analysis was performed by ddPCR assays. A panel of 8 genes (NAMPT, G6PD, GSR, ALDH2, ENO2, PGD, GYS1 and ALDB1H1) was selected among the genes with an adjusted p‐value < 0.05 and a log2FoldChange > |1|. Total RNA was extracted using Direct‐zol RNA Kit (Zymo Research) and reverse transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). The reaction mixture for the ddPCR (20 μl/reaction) contained 2× ddPCR Supermix for Probe (no dUTP) (Bio‐Rad), 20× primer/probe assay for each target (Table S1), 1 ng of cDNA and water up to the final volume. 20 μl of reaction was transferred with 70 μl of Droplet Generation oil for Probes (Bio‐Rad) in a DG8 Cartridge (Bio‐Rad). The cartridges were inserted into the QX200™ Droplet Generator (Bio‐Rad) to generate 40 μl droplet suspension then transferred into a 96‐well PCR plate (Bio‐Rad). The PCR reaction was performed using a GeneAmp™ PCR System 9700 (Applied Biosystems). The protocol conditions consisted of 95°C for 10 min, (94°C for 30 s, 60°C for 1 min) × 40 cycles, 98°C for 10 min and 4 °C for the storage. Amplification signals were read using the QX200™ Droplet Reader and analysed using the QuantaSoft software (Bio‐Rad).

Volani C., Pagliaro A., Rainer J., Paglia G., Porro B., Stadiotti I., Foco L., Cogliati E., Paolin A., Lagrasta C., Frati C., Corradini E., Falco A., Matzinger T., Picard A., Ermon B., Piazza S., De Bortoli M., Tondo C., Philippe R., Medici A., Lavdas A.A., Blumer M.J., Pompilio G., Sommariva E., Pramstaller P.P., Troppmair J., Meraviglia V, & Rossini A. (2022). GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy. Journal of Cellular and Molecular Medicine, 26(13), 3687-3701.

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Detecting TET2 Mutations by ddPCR

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To identify the low-frequency mutation in TET2, ddPCR was performed in RJ-31’s genome DNA at different time points. To make the assay more convinced, water and three healthy controls were included as blank and negative controls, respectively. ddPCR was accomplished on a Bio-Rad QX200 system using ddPCR supermix for Probe (No dUTP) (Bio-Rad) and analyzed on QuantaSoft (Version 1.7.4). The primers and probe sequences targeting TET2 were forward, 5′-GTCCAAGGAGGCTTACACAAAT-3′; reverse, 5′-ACCTCATCGTTGTCCTCTGC-3′; wild probe, 5′VIC-CACACCCTGGACTAG-3′MGB; mutate probe 5′FAM-CACACCCTAGACTAGT-3′MGB. To ensure the quality of the experiment, three replicates were completed for each sample, and each reaction was guaranteed to have a droplet count of more than 10,000.

VAF = \frac{Concentrate of mutate}{Concentrate of mutate and wild}

Yang S., Xu J., Dai Y., Jin S., Sun Y., Li J., Liu C., Ma X., Chen Z., Chen L., Hou J., Mi J.Q, & Chen S.J. (2024). Neutrophil activation and clonal CAR-T re-expansion underpinning cytokine release syndrome during ciltacabtagene autoleucel therapy in multiple myeloma. Nature Communications, 15, 360.

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