SsoAdvanced™ Universal SYBR Green Supermix ® Instruction Manual For use with SYBR® Green–based real-time PCR applications on all real-time PCR instruments Catalog # 172-5270 172-5271 172-5272 172-5274 172-5275
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Table of Contents Sso7d Fusion Enzyme Technology iii Educational Resources iv Reagent Evaluation and Comparison Tutorials iv Protocol 1 Sample Preparation Considerations 1 RNA Samples 1 RNA Integrity and Purity 1 DNA Samples 2 Plasmid Samples 2 Assay Design Considerations 3 Some Key Design Considerations 3 Procedure 4 Reaction Mix Preparation and Thermal Cycling Protocol Real-Time PCR Validation for Gene Expression Experiments 4 5 Determining the Optimal Reference Gene 5 Determin
SsoAdvanced™ Universal SYBR® Green Supermix Catalog # Supermix Volume Kit Size 172-5270 2 ml (2 x 1 ml vials) 200 x 20 μl reactions 172-5271 5 ml (5 x 1 ml vials) 500 x 20 μl reactions 172-5272 10 ml (10 x 1 ml vials) 1,000 x 20 μl reactions 172-5274 25 ml (5 x 5 ml vials) 2,500 x 20 μl reactions 172-5275 50 ml (10 x 5 ml vials) 5,000 x 20 µl reactions Shipping and Storage The SsoAdvanced universal SYBR® Green supermix is shipped on dry ice.
Sso7d Fusion Enzyme Technology Bio-Rad introduced our next generation of real-time PCR supermixes using our patented Sso7d fusion protein technology, delivering a reagent that provides effective performance in a wide range of qPCR applications. The dsDNA-binding protein, Sso7d, stabilizes the polymerase-template complex, increases processivity, and provides greater speed and reduced reaction times compared to conventional DNA polymerases, without affecting PCR sensitivity, efficiency, or reproducibility.
Educational Resources Understanding the Basics To learn more about similarities and differences between PCR and real-time PCR, understand how SYBR® Green and probe-based chemistries function, and see how data are collected and interpreted, please view our interactive tutorial Understanding Real-Time PCR.
Protocol This protocol is intended for use with SYBR® Green-based assays on all real-time PCR systems using a broad range of cycling conditions, template and primer input concentrations, and fast or standard run times.
DNA Samples ■■ ■■ I solate DNA using the appropriate method for the given sample type (for example, column purification for cell lines, phenol/chloroform or column purification for tissue samples) Store the DNA in an appropriate solution – 0.1 mM EDTA (in DEPC-treated ultrapure water) – TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.
Assay Design Considerations When using custom designed assays, several important considerations should be noted: ■■ ■■ ■■ ■■ ■■ ■■ ■■ Biological significance (correct isoform/splice variant chosen) equence quality and secondary structure — evaluate using web-based tools to understand S the complexity of the structure, as it can impact the reaction performance equence length — use the entire gene sequence, or a specific region of interest, to S optimally design an assay equence masking — use web
Procedure Reaction Mix Preparation and Thermal Cycling Protocol 1. Thaw SsoAdvanced™ universal SYBR® Green supermix and other frozen reaction components to room temperature. Mix thoroughly, centrifuge briefly to collect solutions at the bottom of tubes, and then store on ice protected from light. 2. Prepare (on ice or at room temperature) enough reaction setup for all qPCR reactions by adding all required components except the template according to the following recommendations (Table 1). Table 1.
Table 2. Thermal cycling protocol.
4. Perform a reverse transcription reaction for each sample using the same kit, volume, and concentration. Dilute the cDNA, as needed, treating each sample the same to ensure there are no differences from sample to sample in terms of volume and concentration from the initial RNA input. 5. Perform a real-time PCR experiment using the samples and the candidate reference genes using technical triplicates for each sample. 6.
Bio-Rad® iScript™ cDNA Synthesis Kit Serial dilution of the RNA 100 Reaction 1 1 µg RNA 10 –1 Reaction 2 100 ng RNA 10 –2 Reaction 3 10 ng RNA 10 –3 Reaction 4 1 ng RNA 10 –4 Reaction 5 100 pg RNA 10 –5 Reaction 6 10 pg RNA 10 –6 Reaction 7 1 pg RNA Fig. 2. Tenfold serial dilution of RNA starting at 1 µg down to 1 pg, thus covering six logs of dynamic range. Each RNA dilution was transferred to the respective cDNA reaction tube for cDNA synthesis. 4.
6. Evaluate the data. Follow the guidelines in this manual (page 14–15) for setting the baseline and threshold prior to analyzing the data. Figure 4 illustrates the most common results from the experiment and how to interpret the data.
Determining the Real-Time PCR Performance Characteristics Determining the PCR efficiencies of your reference gene and target gene(s) is critical before starting any real-time PCR experiment. Knowing the PCR efficiency determines the appropriate relative gene expression math model. Not knowing may affect and invalidate the results.
3. Cycle according to the recommended protocol. 4. Analyze the data. Follow the guidelines in this manual for setting the baseline and threshold prior to analyzing the data. Tips for Success ■■ ■■ ■■ ipet a minimum of 5 µl for each sample. This ensures greater precision and a smaller standard P deviation for technical replicates. If the samples are too concentrated, simply dilute accordingly.
Dynamic Range Determine the general trend of the slope where linearity (R2) and efficiency are within acceptable ranges, as specified above. Sensitivity Determine the lowest concentration of the serial dilution where replicate reproducibility is high and the R2 of the standard curve is ≥0.980. –d(RFU)/dt Specificity Evaluate the melt profiles when using SYBR® Green–based assays. A single sharp peak with a Tm close to the calculated Tm should be present. 1,200 1,000 800 600 400 200 0 60 70 80 90 Fig.
Troubleshooting Guide Poor Nucleic Acid Yields Review Tables 3 and 4 to determine if you are within an acceptable range of nucleic acid yield. If your yields of RNA are considerably less than is typical for your sample type, reevaluate your isolation method. For reference, typical yields from some mammalian tissues are listed in tables 3 and 4. Table 3. RNA yields. Total RNA per Cell Total DNA per Cell 5–30 pg Varies by genome Table 4. RNA yields per mg of tissue.
Common PCR Inhibitors* From the Sample From the Isolation Method Melanin Polysaccharides Polyphenolics Hemoglobin Chlorophyll Heparin Humic acid Hematin EtOH >1% v/v Proteinase K DMSO >5% EDTA >50 mM SDS >0.01% w/v Sodium Acetate >5 mM Mercaptoethanol Guanidinium Phenol >0.2% v/v DTT >1 mM * Not an inclusive list. 3. If the most concentrated sample in the dilution series is showing compression, as seen in Figure 9, where the tenfold dilution series ∆Cq value is <3.
Low Template Input, Low Expression, High Cq Values If your Cq values are higher than expected or you are concerned about Cq values >30, consider the following corrective actions: 1. Confirm the expected expression level, if known, to ensure that the target of interest is present in your given sample. Additionally, consider higher input concentrations of sample for low expressing targets.
Amplification 16 14 (RFU)(10 ^3) 12 10 8 Cq 6 Cq 8 6 4 2 0 10 0 30 20 Cycles 40 Fig. 11. Baseline setting is best completed in the linear view. In this example, the amplification starts around cycle 8; therefore, setting the end baseline two cycles prior at cycle 6 is best. 2. Either remove this data point or dilute your sample so that it does not show amplification earlier than cycle 15. This ensures that the software’s algorithm has enough background to subtract from the signal.
PCR Performance Not 100% Efficient If you have already ruled out your samples as a source for poor efficiency, then the assay may be the cause of the problem. Please review the section on assay design in this manual for further information (page 3). Also, consider the following corrective action: Perform a temperature gradient experiment to determine the optimal annealing temperature. Set up the gradient as follows: a. Use several representative samples in your project. b.
Table 5. Primer matrix. Forward Primer, nM Reverse Primer, nM 100 150 200 100 100/100 150/100 200/100 150 100/150 150/150 200/150 200 100/200 150/200 200/200 –d(RFU)/dt If your melt profiles exhibit additional peaks at higher melting temperatures than your product of interest (see Figure 14), this is most likely due to nonspecific binding of the primer(s).
If your melt profile exhibits a broad peak, this could be due to the presence of a pseudogene, one product with two melt domains or other target sequence with a similar melting temperature. Due to the nature of SYBR® Green, the lack of ability to discriminate a few base pair differences and very close melting temperatures for two or more amplicons results in this type of melt profile. Consider the following correction action: Melt Peak 500 –d(RFU)/dt 400 300 200 100 0 60 70 80 Temperature, °C 90 Fig.
100 pg Cq 1 ng 10 ng 100 ng 1 µg Initial DNA Fig. 17. Serial dilution of template where the lowest dilution point (100 pg) has lower Cq values than expected due to primer dimer amplification. Control Samples/Wells Are Not Performing as Expected If your non-template control (NTC) wells indicate amplification, you need to determine the source. If primer dimers are not the cause (please review the prior section), then the most likely cause is nucleic acid contamination.
1. Using Table 6, determine the percent of gDNA contamination present. For example, if the ∆Cq (no-RT control Cq – cDNA Cq) for a given sample is seven or greater, then <1% of the DNA present in the sample is gDNA, which would be considered insignificant. Table 6. Determining percent of gDNA contamination. ∆Cq 1 2 3 4 5 6 7 Percent Contribution, % 50.00 25.00 12.50 6.25 3.125 1.5625 0.78125 2. Evaluate the assay design and note the location of the primers.
Ordering Information Catalog # Description SsoAdvanced Universal SYBR Green Supermix 172-5270 2 ml (2 x 1 ml vials), 200 x 20 μl reactions 172-5271 5 ml (5 x 1 ml vials), 500 x 20 μl reactions 172-5272 10 ml (10 x 1 ml vials), 1,000 x 20 μl reactions 172-5274 25 ml (5 x 5 ml vials), 2,500 x 20 μl reactions 172-5275 50 ml (10 x 5 ml vials), 5,000 x 20 µl reactions Two-Step Reverse Transcription Reagents 170-8842 iScript Advanced cDNA Synthesis Kit for RT-qPCR, 50 x 20 μl reactions 170-8843 iScript Advanced
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