| Product Description | Product content: Component | G665836
100 rxns | G665836
100 rxns | G665836
100 rxns | 2×GoldStar Probe One Step Buffer | 1.4 ml | 1.4 ml | 1.4 ml | GoldStar Probe One Step EnzymeMix | 100 μl | 100 μl | 100 μl | 50×Low ROX | - | 50 μl | - | 50×High ROX | - | - | 50 μl | RNase-Free Water | 1.5 ml | 1.5 ml | 1.5 ml |
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Product Introduction：
This product is a specialized reagent kit for one-step Real Time RTqPCR using probe methods (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT qPCR reaction, reverse transcription and quantitative PCR are required
Conducted in the same reaction system, there is no need to add reagents or open the tube cap during the reaction process, avoiding contamination
This has improved the efficiency of the experiment. This product has high detection sensitivity, strong fluorescence signal, and high signal-to-noise ratio, making it very suitable for
Detection of RNA viruses and other trace amounts of RNA. The special buffering system it contains can enable reverse transcriptase to interact with DNA polymerase
Maximize the effectiveness and improve reaction efficiency. By using this product, a wider linear range can be obtained, which is beneficial for the target base Due to more accurate quantification, good repeatability, and high reliability.
ROX dye is used to correct the fluorescence signal error generated between wells in quantitative PCR instruments, and is generally used for ABI
Real Time PCR amplification equipment from companies such as Stratagene. The excitation optical systems of different instruments vary, therefore
The concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.
matters needing attention：
1. Before using the reagents in this reagent kit, please gently mix them upside down to avoid foaming as much as possible, and use them after brief centrifugation. 2. This product uses RNA as a template for one-step RT-PCR experiments, and RNase contamination should be avoided during the operation process,
2.It is recommended to perform RNA operations in a dedicated area, using specialized instruments and consumables. Operators should wear masks and disposable gloves and frequently change gloves. Experimental consumables should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours and sterilized under high pressure for 30 minutes before use.
3. Each reagent in this kit should avoid repeated freezing and thawing as much as possible, as repeated freezing and thawing may lead to a decrease in product performance.
4. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT qPCR reaction. When designing primers, GC content, primer length, and primer should be considered Due to factors such as location, secondary structure of PCR products, it is recommended to use professional primer design software for design.
5. It is recommended to use specific probes in this reagent kit and use professional design software for design.
Usage:
The following examples are typical reaction systems and conditions. In practical operation, corresponding improvements and optimizations should be made based on the differences in template, primer structure, and target fragment size. (Please prepare the reaction solution on ice)
1. Dissolve the RNA template, primers, 2xGoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix, and RNase Free Water and place them on ice for later use.
2. PCR reaction system:
reagent
|
25 μl Reaction system |
final concentration
|
| 2×GoldStar Probe One Step Buffer |
12.5 μl |
1× |
| Forward Primer，10 µM |
0.5 μl |
0.2 μM 1） |
| Reverse Primer，10 µM |
0.5 μl |
0.2 μM 1） |
| Probe ，10 µM |
0.5 μl |
0.2 μM 2） |
| GoldStar Probe One Step EnzymeMix |
1.0 μl |
/ |
| RNA Template |
X μl |
10 pg – 100 ng3） |
| 50×Low ROX or High ROX （optional）4） |
0.5 μl |
1× |
| RNase-Free Water |
up to 25 μl |
/ | |
Note: 1) Typically, the primer concentration is 0.2 μ M can achieve good results, ranging from 0.1 to 1.0 μ M serves as a reference for setting the range. 2) The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.
3) The amount of RNA templates is usually based on 10 pg-100 ng as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the templates to determine the optimal template usage.
4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added. 3. Mix well, centrifuge briefly, and collect the solution to the bottom of the tube.
4. RT-PCR reaction conditions
| steps |
temperature |
time |
/ |
| Reverse Transcription |
45℃ |
10 min |
/ |
PCR pre denaturation
|
95℃ |
10 min |
/ |
denaturation
|
95℃ |
15s |
30-40cycle |
Annealing/Extension
|
60℃ |
45s |
30-40cycle | |
Attention:
1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 5-10 minutes.
2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted. The annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference. |
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