Hifair™ V Multiplex One Step RT-qPCR Probe Kit (UDG Plus) _ 13650ES

YeasenSKU: 13650ES50

Size: 50 T
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Sale price$55.00

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Description

Hifair™ V Multiplex One Step RT-qPCR Probe Kit (UDG Plus) is a multiplex quantitative PCR kit based on RNA as a template. In the process of the experiment, reverse transcription and quantitative PCR were carried out in the same tube, which simplified the experimental operation and reduced the risk of contamination.

In this kit, the first strand cDNA was efficiently synthesized by heat-resistant Hifair™ V Reverse Transcriptase and quantitatively amplified by Unicon™ HotStart Taq DNA Polymerase. The kit mainly contains optimized MP buffer, enzymes mix, etc. The buffer solution already contains Mg2+ and dNTP. In addition, the factors that can effectively inhibit the non-specific PCR amplification and improve the amplification efficiency of multiple qPCR reactions are added, which can ensure the amplification efficiency and carry out up to multiple amplification reactions. The dUTP/UDG system was added to effectively prevent the risk of aerosol contamination.

Features

  • Hot start chemical reagents provide room temperature stability.
  • Complete RT and qPCR steps in a single tube - for convenience and reduced risk of contamination.
  • qPCR was performed using the entire RT reaction as a template -improved sensitivity.

Applications

  • Detection of pathogens
  • Diagnosis and research of tumor
  • Animal disease detection
  • Early diagnosis of hereditary diseases
  • Detection of pathogenic microorganisms in food

Specifications

Hot Start Built-in hot start
Detection method Primer-probe detection
PCR method One step RT-qPCR
Type of sample RNA
Application equipment Equipment with Rox: ABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™, StepOne Plus™ Equipment with Low Rox: ABI 7500, 7500 Fast, ViiA™7, QuantStudio™ 3 and 5, QuantStudio™ 6,7,12k Flex Stratagene MX3000P™, MX3005P™, MX4000P™ Equipment without Rox: Bio-Rad CFX96™, CFX384™, iCycler iQ™, iQ™5, MyiQ™, MiniOpticon™, Opticon®, Opticon® 2, Chromo4™ Eppendorf Mastercycler® ep realplex, realplex 2 s; Qiagen Corbett Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene® 6000 Roche Applied Science LightCycler® 480, LightCycler® 2.0, Lightcycler® 96 Thermo Scientific PikoReal Cycler; Cepheid SmartCycler®; Illumina Eco qPCR

Components

Components

Components No.

Name

13650ES60 

(100 T)

13650ES80 

(1,000 T)

13650ES92 

(10,000 T)

13650-A

2×HifairTM V MP Buffer

1.5 mL

15 mL

150 mL

13650-B

HifairTM V Enzyme Mix

120 μL

1.2 mL

12 mL



Note: 1) 2×Hifair V MP Buffer is the abbreviation of Hifair V Multiplex One Step RT-qPCR Probe Buffer, which includes dNTPs, dUTP, Mg2+, stabilizers, enhancers, and more.

2) Hifair V Enzyme Mix mainly contains heat-resistant Hifair V reverse transcriptase, UNICON HotStart Taq DNA polymeras and UDGase.

 

Instructions

  • Reaction Composition

Components

Volume (μL)

Final Concentration

2×Hifair V MP Buffer

15

Hifair V Enzyme Mix

1.2

-

Primer / Probe Mix (2.5 μM)

3

0.25 μM

Template RNA

1-10

-

RNase Free H2O

to 30

-

Note:Be sure to mix well before use, avoid excessive bubbles caused by violent vibration.

a) Primer concentration: Primer mix including multiplex primer, depending on the situation optimal primer concentration may be between 0.1 and 1.0 μM.

b) Probe concentration: Probe mix including multiplex probe labeling difference fluorescent group, depending on the situation optimal probe concentration may be between 0.05 and 0.5 μM.

c) Template dilution: qPCR is highly sensitive and it is recommended to dilute the template. The control Ct value is suitable between 20 and 35.

d) System preparation: un head with filter element. Avoid cross contamination and aerosol contamination.

    Cycle Setup

    Regular 

    CYCLE STEP
    TEMPERATURE
     TIME  CYCLES
     Reverse Transcription
     50°C*  20 mins
     1
     Initial Denaturation
     95°C  5 mins
     1
     Denaturation
     Extension
     95°C
     60°C
     15 secs
     30 secs
     40-45

     

     Fast 1

    CYCLE STEP
    TEMPERATURE
     TIME  CYCLES
     Reverse Transcription
     50°C*  10 mins
     1
     Initial Denaturation
     95°C  1 min
     1
     Denaturation
     Extension
     95°C
     60°C
     10 secs
     30 secs
     40-45

     

    Fast 2**

    CYCLE STEP
    TEMPERATURE
     TIME  CYCLES
     Reverse Transcription
     50°C*  2 mins
     1
     Initial Denaturation
     95°C  2 secs
     1
     Denaturation
     Extension
     95°C
     60°C
     1 sec
     8 secs
     40-45


    *50°C is optimal for the Hifair™ Reverse Transcriptase. To ensure best performance, avoid using a temperature of < 50°C and >55°C .

    ** “Fast” ramp speed mode can be used where available (e.g., QuantStudio5 and so on).


    Shipping and Storage

    The product is shipped with dry ice and can be stored at -15℃ ~ -25℃ for 1 year. It should avoid repeated freezing and thawing. It is recommended to save separately.

    Figures

    Figure 1 High stability: 37℃ thermal acceleration stability.

    Figure 1 High stability: 37℃ thermal acceleration stability.

    Figure 2 High stability: freeze-thaw stability. 

    Figure 2 High stability: freeze-thaw stability.


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