(96-well) QuickEasy Cell Direct RT-qPCR Kit – SYBR Green I
Descriptions
The (96-well) QuickEasyTM Cell Direct RT-qPCR Kit – SYBR Green I provides a unique lysis buffer system to quickly release RNA from cultured cell samples for RT-qPCR reactions, eliminating the time-consuming and laborious RNA purification process, and only takes 7 minutes to obtain the required RNA template. The 5× Direct RT Mix and 2× Direct qPCR Mix-SYBR provided in the box 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 can use the lysate of the sample to be tested as a template for efficient reverse transcription and specific amplification. The reagent contains Foregene unique Reverse Transcriptase with high affinity for RNA , as well as Hot D- Taq DNA Polymerase , dNTPs, MgCl2 , reaction buffer, PCR optimizer and stabilizer , and can be used in conjunction with the lysis buffer to detect the sample quickly, easily and accurately, and it has the characteristics of high sensitivity, strong specificity and good stability.
The kit is aimed at micro-system lysis of 96-well cultured cells, and has good uniformity and consistency; the kit components provide 96 lysis reactions, 96 reverse transcription reactions and 96×2 qPCR reactions, which can meet the 96-well cells plate for one-time use, avoiding the pollution caused by repeated opening, freezing and thawing of reagents and the degradation of reagent performance.
Kit components
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Kit composition ( 50 μL lysis system/2 0 μL RT reaction system /20 μL qPCR reaction system ) |
DRT-03011 |
Remark |
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96T |
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Part I |
Buffer CL |
5 mL |
Cell Lysis |
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Foregene Protease Plus II |
100 μL |
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Buffer ST |
500 μL |
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Part II |
DNA Eraser |
100 μL |
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5× Direct RT Mix |
400 μL |
RT |
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2× Direct qPCR Mix-SYBR |
1 mL × 2 |
qPCR |
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50× ROX Reference Dye |
400 µL |
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RNase - Free ddH 2 O |
1.7mL |
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Manual |
1 piece |
1 serving |
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*: The lysis reagent DNA Eraser is included in Part II of the kit; Cell Lysis, RT, and qPCR components can be purchased separately.
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.
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-Taqman 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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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 |
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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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qPCR Mix |
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Kit components (20 μl reaction system) |
DRT-01021-C1 |
DRT-01021-C2 |
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200 T |
1000 T |
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2× Direct qPCR Mix-Taqman |
1 ml × 2 |
1.7 ml × 6 |
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20× ROX Reference Dye |
40 μl |
200 μl |
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RNase-Free ddH2O |
1.7 ml |
10 ml |
Instruction Manuals:
