|
The SYBR® Real-Time PCR Kit provides qualified reagents in lyophilized form for the amplification and detection of DNA in quantitative real-time polymerase chain reaction (qPCR). The SuperMix formulation is aliquoted into plate wells or strip wells and then lyophilized for room temperature storage and ease of reaction setup. To perform PCR, simply add water, primers, and template, vortex to dissolve the pellet, and proceed with the reaction.
The system enables highly sensitive detection from as few as 10 copies of a target gene, with a broad dynamic range that supports accurate quantification of high-copy gene from up to 1 μg of DNA:
2×SYBR® Green Reaction Mix consists of a proprietary buffer system, SYBR® Green I, MgSO4, dNTPs, and stabilizers. SYBR® Green I is a fluorescent dye that binds directly to double-stranded DNA (dsDNA). In qPCR, as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and that can be detected using real-time qPCR instruments. SYBR® Green I in this ready to use formulation can quantify as few as 10 copies of a target gene in as little as 1 pg of total DNA or RNA. It has a broad dynamic range of seven orders of magnitude, and is compatible with melting curve analysis.
Uses for the SYBR® Real-Time PCR Kit
Use the One-Step SYBR® Real-Time RT-PCR Kit in your experiments for the following purposes:
-
To quantify a target gene in DNA, especially the low expression genes.
-
To analyze the relative expression ratio between a target gene and a housekeeping gene.
Components of the Kit
This section provides more information about the reagents supplied in the One-Step SYBR® Real-Time RT-PCR Kit.
|
Components
|
Description
|
|
SYBR PCR Master Mix 2×Conc.
|
The 2×SYBR® Green Reaction Mix consists of a proprietary buffer system, SYBR® Green I, MgCl2, dNTPs, and stabilizers. The mix includes 0.2 mM of each dNTP and 3mM MgCl2, and works well for most targets. However, the optimal Mg concentration may range from 3 to 6 mM, and so, if necessary, use the separate tube of 25mM magnesium sulfate to increase the magnesium concentration. It’s very important to store the SYBR PCR Master Mix in the dark.
|
|
Taq DNA polymerase 5U/μl
|
For most PCR reaction, the final concentration of DNA polymerase was usually 0.05U/μl per reaction volume.
|
Performing Real-Time PCR
In SYBR® Green real-time PCR, as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and can be detected using real-time qPCR instruments. This section provides guidelines and an example protocol for performing SYBR real-time PCR.
Instrument Compatibility
This kit can be used with a variety of real-time instruments, including but not limited to the ABI PRISM 7000, 7700, and 7900HT; the ABI 7300 and 7500 Real-Time PCR Systems; the Bio-Rad iCycler; the Stratagene Mx3000P and Mx4000; the Corbett Research Rotor-Gene 3000; the MJ Research DNA Engine Opticon, Opticon 2, and Chromo 4 Real-Time Detector; and the Cepheid Smart Cycler. Optimal cycling conditions will vary slightly with different instruments.
Primer Design & Concentration
Primer selection is one of the most important parameters for qPCR when using a SYBR Green detection system. When designing primers, keep in mind that the amplicon length should be approximately 80–250 bp to optimize the efficiency of qPCR. We strongly recommend using a primer design program such as Oligo 6.0.
Ensure that primers are specific for the target sequence and free of internal secondary structure, and avoid complementation at 3’-ends within each primer and with each other. A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.
Template Specifications
The target template for SYBR real-time PCR is plasmid DNA (10 to 107 copies), genomic DNA (100 pg to 1μg), or cDNA (generated from 1 pg to 100 ng of total RNA). For best results, the amplicon size should be limited to 80–250 bp in size.
Magnesium Concentration
The 2×SYBR Green Reaction Mix includes magnesium at a final concentration of 5 mM which has been confirmed to work well for most targets. However, the optimal concentration may range from 3 to 6 mM. If necessary, use the separate tube of 25mM magnesium chloride to increase the magnesium concentration.
Three-Step Standard Cycling Program
-
95℃ for 3-minute hold;
-
40 cycles of:
-
95℃, 15 seconds
-
55℃, 30 seconds
-
72℃, 30 seconds
-
Melting Curve Analysis
-
Program according to instrument instructions.
Two-Step Standard Cycling Program
-
95℃ for 3-minute hold;
-
40 cycles of:
-
95℃, 15 seconds
-
60℃, 30 seconds
-
Melting Curve Analysis
-
Program according to instrument instructions.
Optimal temperatures and incubation times may vary for different target sequences.
1. Make sure that all components are at the bottom of the tube/plate; centrifuge briefly if needed.
2. See the table on the previous page for the amount/concentration of ROX to use for your specific instrument.
3. Melting curve analysis can identify the presence of primer dimers by their lower annealing temperature, compared to the amplicon. The presence of primer dimers is not desirable in samples that contain template, as it decreases PCR efficiency and obscures analysis and determination of cycle thresholds.
4. The formation of primer dimers most often occurs in no-template controls, where the polymerase enzyme is essentially idle, and in this case the quantitative analysis of the template samples is not affected.
5. Melting curve analysis of no-template controls can discriminate between primer dimers and spurious amplification due to contaminating nucleic acids in reagent components.
Quality Control
The product is tested functionally by qRT-PCR using total HeLa RNA as template. Kinetic analysis must demonstrate a linear dose response with decreasing target concentration and detection of GAPDH mRNA in 1 pg of total HeLa RNA.
Contact Us
For more information or technical assistance, please call, write, fax, or email. Additional international offices are listed on our web page. For technical assistance, email support@genepharma.com. For questions related to order placement, email bd@genepharma.com.
Warranty and Liability:
GenePharma is committed to providing the highest quality reagents at competitive prices. GenePharma warrants that the products meet or exceed the performance standards described in the product specification sheets. If you are not completely satisfied with any product, our policy is to replace the product or credit the full purchase price and delivery charge. No other warranties of any kind, expressed or implied, are provided by GenePharma. GenePharma’s liability shall not exceed the purchase price of the products. GenePharma shall have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products.
ALL PRODUCTS SOLD BY GENEPHARMA ARE INTENDED FOR RESEARCH USE ONLY, UNLESS OTHERWISE INDICATED. THE PRODUCTS ARE NOT INTENDED FOR DIAGNOSTIC OR DRUG PURPOSES.
|
