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Product Information

Product Name

Catalog No.

Size

Hieff Unicon® V Universal Multiplex One Step RT-qPCR Probe Kit

11213ES60

100 T

Hieff Unicon® V Universal Multiplex One Step RT-qPCR Probe Kit

11213ES80

1000 T

Hieff Unicon® V Universal Multiplex One Step RT-qPCR Probe Kit

11213ES92

10000 T

Product Description

Hieff UNICON® V universal multiplex one step RT qPCR Probe Kit 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 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 four reactions.

Contents

Contents No.

Name

11213ES60 (100 T)

11213ES80 (1000 T)

11213ES92 (10000 T)

11213-A

2×HU® MP Buffer

1.25 mL

12.5 mL

125 mL

11213-B

HU® Enzyme Mix

100 μL

1 mL

10 mL

Note

1) 2 × HU® MP buffer is the abbreviation of multiplex one step RT qPCR probe buffer.

2) Enzyme mix mainly contains heat-resistant Hifair® V reverse transcriptase and UNICON® HotStart Taq DNA polymerase.

Applicable 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

Shipping and Storage

The Hieff Unicon® Universal TaqMan multiplex qPCR master mix (UDG plus) is shipped on dry ice. The kit should be stored immediately upon receipt at –20°C in a constant-temperature freezer.

Important points before starting

1. Please use RNase free consumables during the experiment.

2. For your safety and health, please wear lab clothes and disposable gloves.

Reaction composition (for 25 μL)

Component

Volume(μL)

Final Concentration

2×HU® MP Buffer

12.5

HU® Enzyme Mix

1

-

Primer Mix (10 μM)

1 each

0.4 μM

Probe Mix (10 μM)

0.5 each

0.2 μM

Template RNA

1-10 μL

-

RNase Free H2O

to 25 μL

-

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

a) Primer concentration: Primer Mix including multiplex primer, each primer’s final concentration is 0.2 μM, depending on the situation optimal primer concentration may be between 0.1 and 0.5 μM.

b) Probe concentration: Probe Mix including multiplex probe labeling difference Fluorescent group, depending on the situation optimal probe concentration may be between 50 and 300 nM.

c) Template dilution: qPCR has high sensitivity. It is recommended to dilute the template and control CT between 20 and 35.

d) Reaction composition: 25-50 μ L is recommended to ensure the effectiveness and repeatability of target gene amplification.

e) System preparation: Please prepare in the ultra-clean working table, and pipettor and reaction tube without nuclease residue; it is recommended to use the gun head with filter element. Avoid cross contamination and aerosol contamination.

Optimized cycling protocol

Temperature

Time

Cycle

1

Reverse transcription

50 ℃a

10 min

1

2

Initial activation step

95 ℃

5 min

1

3

Amplification

95 ℃

15 sec

45

4

Amplification

60 ℃b

30 secc

45

Note

a) Reverse transcription Temperature: 42℃ or 50℃.

b) Amplification reaction: the reaction temperature was adjusted according to the TM value of designed primers.

c) Fluorescence signal acquisition: different qPCR detection instruments need different fluorescence signal acquisition time, please set according to the shortest time limit.

15221439726

15221439726