Cell Direct RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready One-step qRT-PCR Kits
Descriptions
This kit uses a unique lysis buffer system that can quickly release RNA from cultured cell samples for RT-qPCR reactions, thereby eliminating the time-consuming and laborious RNA purification process. The RNA template can be obtained in just 7 minutes. The 5×Direct RT Mix and 2×Direct qPCR Mix-SYBR reagents provided by the kit can quickly and effectively obtain real-time quantitative PCR results.
5×Direct RT Mix and 2×Direct qPCR Mix-SYBR have strong inhibitor tolerance, and the lysate of the samples can be used as the template for RT-qPCR directly. This kit contains the unique RNA high-affinity Foregene reverse transcriptase, and Hot D-Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR optimizer and stabilizer.
Specifications
200×20μl Rxns, 1000×20μl Rxns
Kit components
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Part I |
Buffer CL |
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Foregene Protease Plus II |
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Buffer ST |
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Part II |
DNA Eraser |
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5× Direct RT Mix |
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2× Direct qPCR Mix-SYBR |
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50× ROX Reference Dye |
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RNase-Free ddH2O |
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Instructions |
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Features&advantages
■ Simple and effective : with Cell Direct RT technology, RNA samples can be obtained in just 7 minutes.
■ The sample demand is small, as low as 10 cells can be tested.
■ High throughput: it can quickly detect RNA in cells cultured in 384, 96, 24, 12, 6-well plates.
■ DNA Eraser can quickly remove released genomes, greatly reduce the impact on subsequent experimental results.
■ Optimized RT and qPCR system makes the two-step RT-PCR reverse transcription more efficient and PCR more specific, and more resistant to RT-qPCR reaction inhibitors.
Kit application
Scope of application: cultured cells.
- RNA released by sample lysis: only applicable to the RT-qPCR template of this kit.
- The kit can be used for the following purposes: gene expression analysis, verification of siRNA-mediated gene silencing effect, drug screening, etc.
Diagram
Storage and Shelf life
Part I of this kit should be stored at 4℃; Part II should be stored at -20℃.
Foregene Protease Plus II should be stored at 4℃,do not freeze at -20℃.
Reagent 2×Direct qPCR Mix-SYBR should be stored at -20℃ in the dark; if used frequently, it can also be stored at 4℃ for short-term storage (use up within 10 days).
Real Time PCR primer design principles
Forward Primer and Reverse Primer
For Real Time PCR, primer design is very important. Primers are related to the specificity and efficiency of PCR amplification, and can be designed with reference to the following principles:
Primer length: 18-30bp.
GC content: 40-60%.
Tm value: Primer design software, such as Primer 5, can give the Tm value of the primer. The Tm values of the upstream and downstream primers should be as close as possible. The Tm calculation formula can also be used: Tm = 4 °C (G + C) + 2 °C (A + T). When performing PCR, a temperature below the primer Tm value of 5 °C is generally selected as the annealing temperature (the corresponding increase in the annealing temperature can increase the specificity of the PCR reaction).
Primers and PCR products:
Design primer PCR amplification product length is preferably 100-150bp.
Design primers in the secondary structural area of the template should be avoided as much as possible.
Avoid the formation of 2 or more complementary bases between the 3′ ends of upstream and downstream primers.
Primer 3′ terminal base can not be present with 3 additional consecutive G or C.
The primers themselves cannot have complementary structures, otherwise a hairpin structure will be formed, affecting PCR amplification.
ATCG should be distributed as evenly as possible in the primer sequence, and the 3′ terminal base should be avoided as T.
Appendix 1：Cell Direct RT-qPCR Kit component supplement pack
1.Cell Lysis Solution
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Kit components (24-well lysis system / well) |
DRT-01011-A1 |
DRT-01011-A2 |
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100 T |
500 T |
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Part I |
Buffer CL |
20 ml |
100 ml |
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Foregene Protease Plus II |
400 μl |
1 ml × 2 |
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Buffer ST |
1 ml × 2 |
10 ml |
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Part II |
DNA Eraser |
400 μl |
1 ml × 2 |
2.RT Mix
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Kit components (20 μl reaction system) |
DRT-01011-B1 |
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200 T |
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5× Direct RT Mix |
800 μl |
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RNase-Free ddH2O |
1.7 ml × 2 |
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Kit components (20 μl reaction system) |
DRT-01011-C1 |
DRT-01011-C2 |
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200 T |
1000 T |
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2× Direct qPCR Mix-SYBR |
1 ml × 2 |
1.7 ml × 6 |
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50× ROX Reference Dye |
40 μl |
200 μl |
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RNase-Free ddH2O |
1.7 ml |
10 ml |
Instruction Manuals:
