Hifair™ V Multiplex One Step RT-qPCR Probe Kit (UDG Plus) _ 13650ES (1G)

SKU: 13650ES50

Size: 50 T
Price:
Sale price$55.00

Shipping calculated at checkout

Stock:
In stock

Description

Hifair™ V Multiplex One Step RT-qPCR Probe Kit (UDG Plus) is a multiplex quantitative PCR kit based on RNA as 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 HifairTM V Reverse Transcriptase and quantitatively amplified by UNICONTM 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 reaction. The dUTP/UDG system was added to effectively prevent the risk of aerosol contamination.

Features

 High sensitivityThe carefully optimized buffer system improves the detection sensitivity of low-concentration templates up to 250 copies/mL

 Super stability: The product has passed real-time stability testing for 18 months..

 dUP/UDG anti-pollution systemdUTP/ UDG anti-pollution system is introduced to degrade aerosol pollution of products efficiently, reduce false positive, and ensure authentic results

 Support fast program: compatible with fast program, 40min can produce results;

Components

Components No.

Name

13650ES50 

(50 T)

13650ES60 

(100 T)

13650ES80

 (1,000 T)

13650ES92

 (10,000 T)

13650-A

2×HifairTM V MP Buffer

750 μL

1.5 mL

15 mL

150 mL

13650-B

HifairTM V Enzyme Mix

60 μL

120 μL

1.2 mL

12 mL


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

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

Storage

The product should be stored at -25°C~-15°C for 18 months.

 

Instructions

Reaction Composition

Components

Volume (μL)

Final Concentration

2×HifairTM V MP Buffer

15

HifairTM V Enzyme Mix

1.2

-

Primer / Probe Mix (2.5 μM)

3

0.25 μM

Template RNA

1-10

-

RNase Free H2O

to 30

-

NoteBe sure to mix well before use, avoid excessive bubbles caused by violent vibration.

a) Primer/Probe concentration: Primer mix including multiplex primer, depending on the situation optimal primer concentration may be between 0.1 and 1.0 μM.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: unhead with filter element. Avoid cross contamination and aerosol contamination.

Optimized Cycling Protocol

 

Reaction stage

Temperature

Time

Cycle

1

Reverse transcription

50°Ca

10 min

1

2

Initial denaturation

95°C

5 min

1

3

Amplification reaction

95°C

15 sec

45 cycles

60°Cb

30 secc

Notea) Reverse transcription: The temperature can select 42°C or 50°C for 10-15 minutes.

b) Amplification reaction: The temperature is adjusted according to the Tm value of the designed primers.

c) Fluorescence signal acquisition: Please set the experimental procedure according to the requirements of the instrument manual.

3. Application equipment

Equipment with RoxABI 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™, StepOne Plus™

Equipment with Low RoxABI 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

Notes

Please wear the necessary PPE, such lab coat and gloves, to ensure your health and safety

Please use RNasefree consumables for the experiment.

Sensitivity test

The 13650 stored at -20°C for 18 months (experimental group) and those within the shelf life (control group) were simultaneously tested for the gradient dilution (500 copies/ml, 250 copies/ml) of the pseudovirus SARS-CoV-2, targeting the ORF gene and N gene. Eight replicate wells were tested for each concentration, with a detection rate of 100%. Moreover, there were no significant differences in the amplification curve peak shapes and Ct values.

Lot

FAM (number of positives)

VIC(number of positives)

250copies/ml

500copies/ml

250copies/ml

500copies/ml

experimental group

8/8

8/8

8/8

8/8

control group

8/8

8/8

8/8

8/8

 

Real-time stability test

The 13650 stored at -20°C for 18 months and those within their shelf life. Both groups were tested simultaneously for the SARS-CoV-2 pseudovirus ORF gene and N gene at a concentration of 1000 copies/ml with 20 replicate wells. The detection rate for both was 100%, and there were no significant differences in the shape of the amplification curves or the Ct values.

 

Figure 1. Red: 13650 within their shelf lifeblue13650 stored at -20°C for 18 months

Stable performance after repeated freeze-thaw cycles

Subjecting the 13650 reagent to 30 and 50 freeze-thaw cycles using dry ice, alongside reagents stored normally at -20°C, and simultaneously testing for the SARS-CoV-2 pseudovirus ORF gene, the results indicated that the performance of the 13650 reagent was unaffected after 50 freeze-thaw cycles.

 

Figure 2. Red: 13650 stored normally at -20°C; Purple13650 to 30 freeze-thaw cyclesGreen13650 to 50 freeze-thaw cycles.

Compatible with Standard and Rapid Protocols

Using 13650ES, the ORF gene and N gene of SARS-CoV-2 pseudovirus were detected under the same reaction system with both standard and rapid protocols. The results showed that under a 4-plex system, there was no difference in the amplification curve peak shapes and Ct values between the two protocols.

 

Documents:

Manuals



Payment & Security

American Express Apple Pay Diners Club Discover Google Pay Mastercard Visa

Your payment information is processed securely. We do not store credit card details nor have access to your credit card information.

Inquiry

You may also like

FAQ

The product is for research purposes only and is not intended for therapeutic or diagnostic use in humans or animals. Products and content are protected by patents, trademarks, and copyrights owned by Yeasen Biotechnology. Trademark symbols indicate the country of origin, not necessarily registration in all regions.

Certain applications may require additional third-party intellectual property rights.

Yeasen is dedicated to ethical science, believing our research should address critical questions while ensuring safety and ethical standards.