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Cell Direct RT qPCR Kit—SYBR GREEN I Direct Cell Lysis Cell Ready One-step qRT-PCR Kits

Kit Description:

Cat.No.DRT-01011/01012

For cell direct RT-qPCR using 10-1,000,000 cells,

and performing RT-qPCR directly from cells without prior RNA purification.

Cells are lysed directly to release RNA for RT-qPCR; the high tolerance system makes it unnecessary to purify RNA and directly use cell lysates as RNA templates for RT reactions. Fast and convenient; high sensitivity, strong specificity, and good stability.

◮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.


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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

Part I

Buffer CL

Foregene Protease Plus II

Buffer ST

Part II

DNA Eraser

5× Direct RT Mix

2× Direct qPCR Mix-SYBR

50× ROX Reference Dye

RNase-Free ddH2O

Instructions

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

Cell Direct RT qPCR 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 -20in the dark; if used frequently, it can also be stored at 4℃ for short-term storage (use up within 10 days).


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  • 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


    Cell Lysis Solution

    Kit components

    (24-well lysis system / well)

    DRT-01011-A1

    DRT-01011-A2

    100 T

    500 T

    Part I

    Buffer CL

    20 ml

    100 ml

    Foregene Protease Plus II

    400 μl

    1 ml × 2

    Buffer ST

    1 ml × 2

    10 ml

    Part II

    DNA Eraser

    400 μl

    1 ml × 2

    2.RT Mix


    RT Mix

    Kit components

    (20 μl reaction system)

    DRT-01011-B1

    200 T

    5× Direct RT Mix

    800 μl

    RNase-Free ddH2O

    1.7 ml × 2

     

    3.qPCR Mix


    qPCR Mix

    Kit components

    (20 μl reaction system)

    DRT-01011-C1

    DRT-01011-C2

    200 T

    1000 T

    2× Direct qPCR Mix-SYBR

    1 ml × 2

    1.7 ml × 6

    50× ROX Reference Dye

    40 μl

    200 μl

    RNase-Free ddH2O

    1.7 ml

    10 ml

    Instruction Manuals:

    QuickEasyTM Cell Direct RT-qPCR Kit-SYBR Green I

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