RT-QPCR EasyTM (One Step)-Taqman Cat.No.RT-02131/02132 One-Step Real-Time RT-PCR Master Mix

Cat.No.RT-02131/02132

One-step Real-Time RT-PCR Master Mix

RT-PCR EasyTM I(One Step)

Cat.No.RT-02011/02012

One-step RT-PCR Master Mix

For research use only

Store at -20℃

Product Introduction

Foregene One-Step RT-PCR EasyTM series products realize the integrated reaction from RNA to double-stranded DNA, that is, reverse transcription and PCR amplification are completed in the same reaction centrifuge tube and the same reaction system, which simplifies the experimental steps, optimizes the experimental program, and improves the experiment efficiency.

RT-qPCR EasyTM (One Step)-Taqman using Foregene HotStar Taq DNA Polymerase, the optimized Taqman qPCR system can quickly and specifically perform Real Time RT-qPCR quantitative detection of trace RNA templates.

Features

- One-step kit enables reverse transcription and PCR to be carried out in the same tube, only need to add template RNA, specific PCR primers and RNase-Free ddH2O.

- Real-time quantitative analysis of viral RNA or trace RNA can be carried out quickly and accurately.

- The kit uses a unique Foregene reverse transcription reagent and Foregene HotStar Taq DNA Polymerase combined with a unique reaction system to effectively improve the amplification efficiency and specificity of the reaction.

- The optimized reaction system makes the reaction have higher detection sensitivity, stronger thermal stability, and better tolerance.

- RT-qPCR EasyTM (One Step)-Taqman kit comes with ROX internal reference dye, which can be used to eliminate signal background and signal errors between wells, which is convenient for end users to use in different models of quantitative PCR instruments

The control of product quality

According to FOREGEN's Total Quality Management System (FOREGENE's Total Quality Management System), each batch of RT-PCR Easy^TM I (One Step) kits are strictly tested multiple times to ensure the quality,reliability and stability of each kits batch .

Kit Contents

Transport and storage conditions

1.Transportation conditions

The whole process of low-temperature ice box transportation, to ensure that the kit is in a state of <4 °C.

2. Storage conditions

RT EasyTM I is stored at -20°C. Store the product in a constant temperature refrigerator at -20°C immediately after receipt. If the storage conditions are appropriate, the product will not degrade any performance during the 1-year validity period.

Kit component information

2× RT-PCR EasyTM Mix:Foregene Reverse Transcriptase, RNase Inhibitor, Foregene HotStar Taq DNA Polymerase,dNTPs, Mg2+,reaction buffer, optimizer and stabilizer, etc.

Precautions: (Please read the precautions carefully before using the kit)

For templates, it is recommended to use RNA extracted from fresh samples or stored at -80℃ (RNA should avoid repeated freezing and thawing).

In order to avoid RNase contamination, the experiment operation should be carried out in the RNase-Free space; the pipette tips and PCR centrifuge tubes used must be RNase-Free; and disposable gloves and masks should be worn. Before use, put the 2×RT-PCR EasyTM Mix on ice to completely melt, flick and mix well before use; the preparation of the system should be operated on an ice bath to improve the performance of the kit and the specificity of PCR amplification.

Template RNA concentration

RT-PCR EasyTM I(One Step):(0.1pg-1μg total RNA)/20μl system

Kit Application

- This kit is used for Rapid detection of high-copy and low-copy genes.

- It is especially suitable for rapid detection of RNA templates with high GC content or complex secondary structure.

The control of product quality

According to FOREGEN's Total Quality Management System (FOREGENE's Total Quality Management System), each batch of RT-PCR Easy^TM I (One Step) kits are strictly tested multiple times to ensure the quality,reliability and stability of each kits batch .

Operation guide

A: Preparation of materials and reagents

1. Prepare the prepared RNA template (it is recommended to use Foregene Total RNA Isolation Kit series kits to extract and purify RNA), specific primers (10μM) and related consumables and instruments.

Note: Please ensure the integrity of RNA and try to use RNA extracted from fresh samples.

2. Place 2× RT-PCR EasyTM Mix and RNase-Free ddH2O on an ice bath to let it melt naturally, and flick the tube wall to mix well for later use.

B: RT-PCR system preparation

2×RT-PCR EasyTM Mix is convenient and quick to use, avoiding pollution during operation and experimental errors caused by multiple preparations of the reaction system to the greatest extent. When using, only half the volume of the reaction system (for example, if the reaction system is 20μl, take 10μl 2×RT-PCR EasyTM Mix solution), add appropriate amount of RNA template and specific primers, and add RNase-Free ddH2O to make up the volume. Refer to Table 1 below for the specific RT-PCR reaction system preparation.

Form 1:RT-PCR system preparation

1*: Usually the final concentration of primer is 0.2-0.25μM to get better results. When the reaction performance is poor, the primer concentration can be adjusted within the range of 0.1-0.5μM.

Note: Forward Primer and Reverse Primer are the specific primers for the target gene; 50μl system, please refer to the 20μl system to adjust the reagent dosage proportionally.

C: RT-PCR reaction program setting

• After preparing the RT-PCR system with reference to the above table, mix gently (you can use pipette tip to blow gently; you can also mix on a vortexer and centrifuge briefly to collect the liquid scattered on the tube wall or tube cap, and place on ice box for later use).

2. Refer to the RT-PCR reaction program settings (Table 2) to set the temperature and time of the reaction.

Note: In order to ensure the activity of 2×RT-PCR EasyTM Mix and improve its amplification efficiency, it is best to prepare the RT-PCR reaction system after setting the PCR instrument program, so that the reaction program can be entered immediately after the system is prepared.

Form 2:RT-PCR reaction system setting

Note: The above procedure is for reference only. The actual reaction conditions vary depending on the structural conditions of the template, primers, etc. For RNA templates with complex secondary structures, the reaction temperature for the first step of reverse transcription is recommended to be 50°C. In the specific operation, it is necessary to design the optimal reaction conditions, including annealing temperature, extension time, etc. according to the specific conditions of the target fragment size, the base sequence of the amplified fragment, and the GC content and length of the primer.
RT-PCR Operation Diagram

Foregene Reverse Transcriptase

Foregene reverse transcriptase can provide highly efficient and specific reverse transcription reaction. Reverse transcriptase exhibits high affinity and still maintains good reverse transcription activity at a reaction temperature of 50°C. It can well reverse transcribe RNA with complex secondary structure that other reverse transcriptases cannot handle. Foregene Reverse Transcriptase has high sensitivity and is applicable in a wide range of RNA amounts (0.1pg≤RNA≤1μg).

Foregene HotStar Taq DNA Polymerase

Provides a highly specific amplification of PCR. When reverse transcription is in progress, DNA polymerase is completely ineffective and does not hinder the reverse transcription reaction. After the PCR reaction program, heated to 94°C, the DNA polymerase is activated and the reverse transcriptase is inactivated. The hot-start process eliminates the formation of non-specific annealing primers and primer dimers in the first cycle, ensuring high specificity and reliability of PCR amplification.

Caution:(Please read the precautions carefully before using the kit)

- Reagents should avoid repeated freezing and thawing, otherwise the performance of the reagents will decrease or become invalid.

- It is recommended to use fresh sample extraction or template RNA stored at -80℃ (RNA should avoid repeated freezing and thawing).

- In order to avoid RNase contamination, the experiment operation should be performed in the RNase-Free space; the pipette tips and PCR tubes used must be RNase-Free; and disposable gloves and masks should be worn.

- This kit must be used with specific primers for experiments. Please select the specific primers for the gene to be amplified according to the needs of the experiment.

- Before use, put 2× RT-PCR Easy^TM Mix on ice to completely melt, flick and mix well before use; the preparation of the system should be operated on an ice bath to improve the performance of the kit and the specificity of PCR amplification .

- Ice bath

Self-prepared reagents

- RNA template

- Gene specific PCR primers

Safety

       - This product is for scientific research use only, please do not use it in medicine, clinical medicine, food and cosmetics. 

       - Wear suitable laboratory clothes, gloves, protective glasses, etc. when using chemicals.

The negative control has too high fluorescence value

1. Reagent contamination caused during operation.

Recommendation: Replace with new reagents for RT-PCR experiments.

2. Contamination occurred during the preparation of the PCR reaction system. Recommendation: Take necessary protective measures during operation, such as: wearing latex gloves, using a pipette tip with a filter, etc.

3. The primers are degraded, and the degradation of the primers will lead to non-specific amplification.

Suggestion: Use SDS-PAGE electrophoresis to detect whether the primers are degraded, and replace them with new primers for RT-PCR experiments.

Poor repeatability of quantitative values

1. The instrument is malfunctioning.

Suggestion: There may be errors between each PCR hole of the PCR machine, resulting in poor reproducibility during temperature management or detection.

Please carry out the test according to the instructions of the corresponding instrument.

2. The sample purity is not good.

Recommendation: Impure samples will lead to poor reproducibility of the experiment, which includes the purity of the template and primers. It is best to repurify the template, and the primers are best purified by SDS-PAGE.

3. The PCR system preparation and storage time is too long.

Suggestion: Use the RT-PCR system for PCR experiments immediately after preparation, and do not leave it for too long; it is recommended to set up the PCR program before proceeding with the RT-PCR system preparation.

4. The PCR amplification conditions are not suitable, and the primer sequence or concentration is improper.

Suggestion: confirm the correctness of the primer sequence and the primer has not been degraded; when the amplification signal is not good, try to adjust the annealing temperature and adjust the primer concentration appropriately.