Ssofast master mix

Manufactured by Bio-Rad

Sourced in United States

SsoFast master mix is a ready-to-use, 2x concentrated, and highly sensitive PCR reaction mix designed for fast, efficient, and reliable real-time PCR. It contains a hot-start DNA polymerase, optimized buffer, and dNTPs.

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Close spelling variants of this product

SsoFast EvaGreen Master Mix (23 citations) SsoFast SYBR Green Master Mix (4 citations) Ssofast PCR master mix (3 citations) SsoFast Evergreen PCR master mix (2 citations) SsofastTM Evagreen (SYBR Green) qPCR master mix (2 citations)

9 protocols using ssofast master mix

Quantifying Brain TRPM2 and AR Expression

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TRPM2 mRNA expression was analyzed in nonsurgical hormonally intact adult males and females (8-12 weeks). Androgen receptor expression was analyzed in contralateral cortex at 24 h after MCAO. Animals were decapitated under isoflurane anesthesia, brains were rapidly removed, and cortices were isolated and rapidly frozen. Total RNA was isolated from approximately 10 mg of cortical tissue (RNAqueous-4 PCR kit; Ambion) and eluted RNA was treated with DNAse (Ambion). First-strand cDNA was synthesized from 500 ng total RNA by reverse transcription using High Capacity cDNA Archive Kit (Applied Biosystems). Quantitative real-time polymerase chain reactions (qRT-PCR) were run in triplicate using SsoFast Mastermix (Biorad) on a Biorad CFX Connect real-time PCR system. FAM-labeled primer/probes used to detect mRNA transcripts of 18S, TRPM2, and the androgen receptor were synthesized by Applied Biosystems. Expression levels were calculated using the ΔΔC_T method relative to the internal control, 18S, and were normalized to male expression levels.

Quillinan N., Grewal H., Klawitter J, & Herson P.S. (2014). Sex Steroids Do Not Modulate TRPM2-Mediated Injury in Females following Middle Cerebral Artery Occlusion,,. eNeuro, 1(1), ENEURO.0022-14.2014.

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Total RNA Extraction and qPCR Analysis

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Total RNA was isolated from cells using RNeasyMinikit (Qiagen, Chatsworth, CA) according to the manufacturer's protocol. Then 1 µg RNA was converted to cDNA using a First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Real-time quantitative PCR reactions were set up in triplicate with Ssofast Master Mix (Biorad, Hercules, CA) and run on a LightCycler® 480 (Roche, Indianapolis, Indiana).

Zhang L., Kim S.B., Jia G., Buhemeida A., Dallol A., Wright W.E., Fornace A.J., Al-Qahtani M, & Shay J.W. (2015). Exome Sequencing of Normal and Isogenic Transformed Human Colonic Epithelial Cells (HCECs) Reveals Novel Genes Potentially Involved in the Early Stages of Colorectal Tumorigenesis. BMC Genomics, 16(Suppl 1), S8.

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Quantitative PCR Analysis of Gene Expression

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QPCR was performed on a CFX-96 thermocycler using SsoFast Master Mix (Bio-Rad) and TaqMan primer-probe sets for each gene (Applied Biosystems) and the data analyzed using the standard curve method. Expression of beta-D-glucuronidase (GUSB) was used to normalize for variances in input cDNA. For all experiments, each sample was run in triplicate wells on each plate and averaged, and each gene was analyzed in triplicate plates.

DeFilippis R.A., Fordyce C., Patten K., Chang H., Zhao J., Fontenay G.V., Kerlikowske K., Parvin B, & Tlsty T.D. (2014). Stress Signaling from Human Mammary Epithelial Cells Contributes to Phenotypes of Mammographic Density. Cancer research, 74(18), 5032-5044.

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Quantitative PCR analysis of GLA transgene expression

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Total RNA was purified from homogenized liver tissue using the QIAGEN AllPrep DNA/RNA Mini Kit, and cDNA was synthesized using the QIAGEN QuantiTect Reverse Transcription kit, according to the manufacturer’s protocol. qPCR was performed using the SsoFast master mix (Bio-Rad) and a custom primer/probe mix targeting the ST-920PC GLA cDNA transgene (forward primer 5′- CGTTGAAAGACCTGCTGTAATC-3′, reverse primer 5′-CAGATGGCTGGCAACTAGAA-3′, and probe 5′-CTGCAGGAATTCGGCTCGAGATCC-3′). Serially diluted linearized ST-920PC plasmid DNA (1.0E+6, 2.5E+5, 6.25E+4, 15,625, 3,906.25, 976.56, 244.14, or 61.04 copies) was used to generate a standard curve. mRNAs from four AAV2/6-hGLA-treated mice (one from each dose group) were directly added to PCRs, without first performing the RT step, as negative controls.

Yasuda M., Huston M.W., Pagant S., Gan L., St. Martin S., Sproul S., Richards D., Ballaron S., Hettini K., Ledeboer A., Falese L., Cao L., Lu Y., Holmes M.C., Meyer K., Desnick R.J, & Wechsler T. (2020). AAV2/6 Gene Therapy in a Murine Model of Fabry Disease Results in Supraphysiological Enzyme Activity and Effective Substrate Reduction. Molecular Therapy. Methods & Clinical Development, 18, 607-619.

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RNA Extraction and qPCR Analysis

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Before collecting RNA, cells were rinsed with Dulbecco’s PBS (Thermo Fisher Scientific). RNA was collected and purified using RNAqueous or RNeasy RNA extraction kits (Thermo Fisher Scientific and Qiagen). After isolation, potential genomic DNA was removed by Turbo DNAse (Thermo Fisher Scientific) or DNAse (Qiagen) as per manufacturer’s directions. cDNA was synthesized from 1 mg of total RNA using the SuperScript^® III First-Strand Synthesis SuperMix for qRT-PCR (Thermo Fisher Scientific). cDNA was diluted 1:30 in water to be in the middle of the five-fold dilution range for 100% efficiency.
QPCR was performed using Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) on the Step One (Applied Biosystems) or SSO Fast Master mix (Biorad) on the CFX96 Connect Real Time System (Biorad) with the following cycling parameters: Initial 95°C for 30 seconds, then 40 cycles of 95°C for 5 seconds and 60°C for 10 seconds. Taqman Probes (Thermo Fisher Scientific) were as follows in Table 1. QPCR data analysis and calculation of error bars was completed using the software and standard settings provided by the manufacturer of the Step One Software (Applied Biosystems) and CFX Manager Software (Biorad). Relative mRNA levels were determined by comparing naïve hESCs using the ΔΔCt method [18 ].

McCabe K.L., Kunzevitzky N.J., Chiswell B.P., Xia X., Goldberg J.L, & Lanza R. (2015). Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation. PLoS ONE, 10(12), e0145266.

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High-throughput qPCR on Fluidigm BioMark IFC

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qPCR was performed on a BioMark 48.48 Integrated Fluidic Circuit (IFC) (Fluidigm, South San Francisco, USA) combining 48 pre-amplified samples with 48 pairs of primers for multi-parallel qPCR reactions. The sample mix included: SSoFast Master Mix (Bio-Rad, Hercules, CA, USA) and 20× sample loading reagent (Fluidigm, South San Francisco, CA, USA) for each pre-amplified cDNA. Each of the 88 assay mixes was prepared with 100 μM of the respective primer pair and DNA suspension buffer (Buffer TE). Thermal cycling and detection were conducted using a BioMark instrument (Fluidigm, South San Francisco, CA, USA) with the following conditions: 15 min at 95 °C, 30 cycles at 94 °C for 10 s, 54 °C for 30 s, and 72 °C for 10 s. Melt curve analysis (70–90 °C) was performed following PCR amplification to assess each amplicon. Cq values and amplification curves were analyzed using Real-Time PCR Analysis software 4.5.2 (Fluidigm, South San Francisco, CA, USA).

Rosas-García J., Ramón-Luing L.A., Bobadilla K., Meraz-Ríos M.A., Sevilla-Reyes E.E, & Santos-Mendoza T. (2022). Distinct Transcriptional Profile of PDZ Genes after Activation of Human Macrophages and Dendritic Cells. International Journal of Molecular Sciences, 23(13), 7010.

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Purkinje Cell Transcriptional Profiling

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Cerebellums from control animals were rapidly removed and snap frozen in OCT cryoprotective media and stored at -80°C until sectioning. Sections (8 μm) were sectioned using a cryostat, collected on uncharged slides and stored on dry ice. Laser capture microdissection (LCM) was performed on the same day as sectioning. Sections were ethanol fixed (70%), dehydrated in alcohol containing RNase inhibitor (Protect RNA 500x concentrate, Sigma-Aldrich, St. Louis, MO) and cleared with xylene. LCM was performed using an ArcturusXT microdissection system (Arcturus, New York, NY). Purkinje cells were captured with the spot tool to select individual cells to be collected on HS LCM microcaps using an infrared laser. Caps were pooled to yield 800-1000 cells from each animal. RNA extraction and isolation was performed using Arcturus PicoPure Kit. cDNA was generated from 100 ng RNA using iScript cDNA synthesis kit (BioRad, Hercules, CA). Quantitative RT-PCR was performed on CFX96 real-time PCR system (BioRad) using taqman primer/probes (Invitrogen, Grand Island, NY) for 18s, GluN1, GluN2A, GluN2A, GABA_A α1 and GABA_A α6 and SsoFast master mix (BioRad). Relative expression was determined using the 18s reference gene and the ΔΔC_q method and data were normalized to GluN1 expression.

Quillinan N., Grewal H., Deng G., Shimizu K., Yonchek J.C., Strnad F., Traystman R.J, & Herson P.S. (2014). Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia. Neuroscience, 0, 555-565.

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Comprehensive RNA Profiling Protocol

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Total RNA was isolated from cell lysates or mouse tissue using an RNeasy Plus Universal Mini Kit (Qiagen) according to the manufacturer’s protocol. Then, 1 μg RNA was converted to cDNA using a First Strand cDNA Synthesis Kit (Roche). Real-time quantitative PCR reactions were set up in triplicate with Ssofast Master Mix (Bio-rad) and run on a LightCycler 480 (Roche). Restriction fragment length polymorphism analysis for the detection of the mutant Kras was performed as described previously (55 (link)). All the primers (Sigma) used in this study are listed in tables S3 and S4.

Zhang L., Theodoropoulos P.C., Eskiocak U., Wang W., Moon Y.A., Posner B., Williams N.S., Wright W.E., Kim S.B., Nijhawan D., De Brabander J.K, & Shay J.W. (2016). Selective targeting of mutant adenomatous polyposis coli (APC) in colorectal cancer. Science translational medicine, 8(361), 361ra140.

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Quantitative PCR analysis of gene expression

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RNA was collected using a PureLink RNA Mini Kit (Life Technologies) and converted to cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was conducted in a Bio-Rad CFX96 real-time PCR system using Applied Biosciences TaqMan primers and probes and Bio-Rad SsoFast master mix. Analysis was conducted using Bio-Rad CFX Manager software, and message amounts were normalized to glyceraldehyde-3-phosphate dehydrogenase.

Hatakeyama J., Wald J.H., Rafidi H., Cuevas A., Sweeney C, & Carraway KL I.I.I. (2016). The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3. Science signaling, 9(434), ra65.

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