Ssofast sybr green master mix

Manufactured by Bio-Rad

Sourced in Canada

The SsoFast SYBR Green Master Mix is a ready-to-use reaction mix for quantitative PCR (qPCR) applications. It contains a fast-cycling Sso7d-fusion polymerase, SYBR Green I dye, and optimized buffer components.

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Lab products found in correlation

Turbo dnase, by Thermo Fisher Scientific (2 mentions) Iscript cdna synthesis kit, by Bio-Rad (2 mentions) Pcr cycler, by Eppendorf (1 mentions) Fluidigm 96.96 dynamicarray ifc, by Standard BioTools (1 mentions) Superscript 3 taq polymerase, by Thermo Fisher Scientific (1 mentions) Exonuclease 1, by New England Biolabs (1 mentions) Biomark hd system, by Standard BioTools (1 mentions) Prism 8, by GraphPad (1 mentions) Cfx96 pcr machine, by Bio-Rad (1 mentions) Tri reagent, by Merck Group (1 mentions) Powerplant rna isolation kit, by Qiagen (1 mentions) Cfx manager, by Bio-Rad (1 mentions)

4 protocols using ssofast sybr green master mix

qRT-PCR Validation of Alfalfa Housekeeping Genes

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For qRT-PCR validation, RNA was treated with TURBO DNase (Ambion, Austin, TX). A total of 1 µg RNA was used to synthesize cDNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Mississauga ON). qRT-PCR amplification was conducted using a C1000 Touch™ Thermocycler Real-Time PCR System (Bio-Rad, Canada) using SsoFast SYBR Green Master Mix (Bio-Rad Laboratories, Mississauga, ON). Alfalfa homologues for two well-known housekeeping genes, ubiquitin (Medtr3g112230) and elongation factor (Medtr1g101870) with little variation of expression in our RNA-seq study, were used as reference genes for qRT-PCR reactions. Gene-specific primers and primers for reference genes are listed in Supplementary Table 4.

Arshad M., Gruber M.Y, & Hannoufa A. (2018). Transcriptome analysis of microRNA156 overexpression alfalfa roots under drought stress. Scientific Reports, 8, 9363.

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Single-cell gene expression profiling

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For gene expression analyses, pools of 100 cells were sorted into wells of a DNase and RNase-free 96-well plate (Applied Biosystems) containing 5 μl CellsDirect 2x reaction buffer (Invitrogen), centrifuged for 5 minutes at 500×g, snap-frozen on dry ice and stored at −80°C until use. RNA was reverse-transcribed using Superscript III Taq polymerase (Invitrogen) and preamplified for 18 rounds with a custom 96-target DeltaGene (Fluidigm) primer panel on a PCR cycler (Eppendorf). Excess primers were removed from the preamplified product by incubating with Exonuclease-1 (New England Biolabs), and cDNA samples were diluted in DNA buffer. Primers and cDNAs mixed with SsoFast Sybr Green Master Mix (BioRad) were subsequently loaded onto a Fluidigm 96.96 Dynamic Array IFC and run on a BioMark HD system (Fluidigm). Data were subsequently analyzed using Fluidigm Gene Expression Software and normalized to Gusb. Relative changes were subsequently calculated using ΔΔCt approach. Unsupervised clustering of Gusb-normalized delta CT values with Gusb removed along with poorly performing Ebf1 and Hoxa2 primer sets, was performed using average linkage. Clustering and principal component analysis (PCA), and heatmap generation, were performed using ClustVis software (biit.cs.ut.ee/clustvis). PCA and PCA loading plots were generated using Prism 8 (Graphpad) from data generated by ClustVis.

Rabe J.L., Hernandez G., Chavez J.S., Mills T.S., Nerlov C, & Pietras E.M. (2019). CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions. Experimental hematology.

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Quantitative Real-Time PCR Protocol

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Total RNA was extracted by using Tri-reagent (Sigma-Aldrich). Real-time PCR was performed on a CFX-96 PCR machine (BioRad) using the SsoFastSYBR™ green master mix (BioRad) and following the manufacturer’s guidelines. The primer sequences for measuring all of our genes of interest were purchased from NZYTech and are shown in Table S1 in Additional file 3. Data was analysed as described in [29 (link)] using the 2^-ΔΔCT methodology.

Hill R., Kalathur R.K., Callejas S., Colaço L., Brandão R., Serelde B., Cebriá A., Blanco-Aparicio C., Pastor J., Futschik M., Dopazo A, & Link W. (2014). A novel phosphatidylinositol 3-kinase (PI3K) inhibitor directs a potent FOXO-dependent, p53-independent cell cycle arrest phenotype characterized by the differential induction of a subset of FOXO-regulated genes. Breast Cancer Research : BCR, 16, 482.

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Quantitative RT-PCR Analysis of Gene Expression

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Leaf and root samples were ground using mortar and pestle into fine powder and 100 mg representative sample was used for RNA extraction using the PowerPlant RNA Isolation Kit (Mo Bio Laboratories Inc., San Diego, CA, USA). Extracted RNA was treated with TURBO DNase (Ambion, Austin, TX, USA). One microgram of total RNA was used for cDNA synthesize using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Mississauga, ON, Canada). Gene amplification was performed in a C1000 Touch^TM Thermocycler Real-Time PCR System (Bio-Rad) using SsoFast SYBR Green Master Mix (Bio-Rad). QRT-PCR conditions were 1 cycle at 95°C for 30 s, then 40 cycles at 95°C for 5 s, 60°C for 15 s, followed by melting curve 65–95°C with 5 s/step, +0.5°C/cycle. QRT-PCR data was analyzed using a CFX-Manager (Bio-Rad) with a 2^-ΔCT method (Schmittgen and Livak, 2008 (link)). Suitable reference genes (ACC1, Actin) were selected (Supplementary Figure 3) and primers for reference and target genes were designed from M. sativa sequences (Gao et al., 2016 (link)) and are listed in Supplementary Table 1.

Arshad M., Gruber M.Y., Wall K, & Hannoufa A. (2017). An Insight into microRNA156 Role in Salinity Stress Responses of Alfalfa. Frontiers in Plant Science, 8, 356.

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