Description

RT-qPCR EasyTM (One Step)-SYBR Green Iuses a unique system to effectively combine RT and Real Time PCR reactions, which can quickly complete the quantitative detection of genes. The product has strong tolerance, high specificity and stability. The product contains the company's unique Foregene HotStar Taq DNA Polymerase, which has the advantages of faster amplification (2Kb/min), high amplification efficiency, strong specific amplification ability, and low mismatch rate compared with ordinary Taq enzymes. It is used here for Real Time RT-PCR to reduce non-specific amplification and improve the accuracy of PCR.

Features

One-step kit makes reverse transcription and qPCR two reactions in the same tube, only need to add template RNA, specific PCR primers and RNase-Free ddH2O.
The kit can quickly and efficiently quantitatively analyze viral RNA or trace RNA.
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)-SYBR Green I kit comes with ROX internal reference dye, which can be used to eliminate signal background and signal errors between wells, which is convenient for customers to use in different models of quantitative PCR instruments.

Kit Contents

Kit components	RT-02111	RT-02112
Kit components	100T (20μl system)	500T (20μl system)
2× RT-qPCR EasyTM Mix-SYBR	1ml	1.7ml×3
50× ROX Reference Dye	200μl	1ml
RNase-Free ddH2O	1.7ml	1.7ml×3
Instruction Manual	1 piece	1 piece

Kit component information

2× RT-qPCR EasyTM Mix-SYBR:Foregene Reverse Transcriptase, RNase Inhibitor, Foregene HotStar Taq DNA Polymerase, optimized ratio of dNTPs, SYBR GreenI, Mg2+, stabilizers, enhancers, and optimizers.

ROX Reference Dye:it is generally used on Real Time PCR amplifiers of companies such as ABI and Stratagene to adjust the difference between PCR tubes and tubes caused by PCR sample loading errors. The concentration of ROX Reference Dye required by different instruments is different, and the user can add it according to the recommended concentration of the instrument.

Precautions: (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.

The reagents in the kit should be protected from light and stored at -20°C.

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

In order to avoid RNase contamination, please perform the experiment in the RNase-Free space; the pipette tips and PCR tubes used must be RNase-Free; and wear disposable gloves and masks.

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-qPCR EasyTM Mix-SYBR I 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 increase PCR amplification The specificity.

2×RT-qPCR EasyTM Mix-SYBR contains SYBR Green I, please avoid strong light.

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

The kit is stored at -20℃. Store the product in a constant temperature refrigerator at -20℃ immediately after receipt. If the storage conditions are appropriate, the product will not degrade any performance during the 1-year validity period.
2×RT-qPCR EasyTM Mix-SYBR component contains SYBR Green I, please keep it away from light.

Template RNA concentration

RT-qPCR EasyTM (One Step)-SYBR Green I:(0.1pg-100ng total RNA)/20μl system.

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. Put 2×RT-qPCR EasyTM Mix-SYBR, RNase-Free ddH2O, and 50×ROX Reference Dye (if necessary) on ice to let it melt naturally, and flick the tube wall to mix until use.

B:RT-qPCR system preparation

2× RT-qPCR EasyTM Mix-SYBR is convenient and quick to use, and avoids 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-qPCR EasyTM Mix-SYBR solution), add RNA template and specific primers, and add RNase-Free ddH2O to make up the volume. Refer to Table 1 below for the specific RT-qPCR reaction system preparation

Form 1:RT-qPCR system preparation

RT-qPCR system additions	Amount	Final concentration
2× RT-qPCR EasyTM Mix-SYBR	10μl	1×
Forward Primer (10μM)	0.5μl	0.2-0.25μM 1*
Reverse Primer (10μM)	0.5μl	0.2-0.25μM 1*
Template(RNA)	Xμl	0.1pg-100ng
50× ROX Reference Dye	-	2*
RNase-FreeddH2O	(9-X)μl
Total Volume	20μl

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 specific primers for the target gene.

2: Choose the appropriate final concentration of ROX Reference Dye according to different quantitative PCR instruments. The optimal concentration of ROX Reference Dye for common quantitative PCR machines is shown in the following table:

Quantitative PCR instrument	ROX Reference Dye final concentration
ABI PRISM 7000/7300/7700/7900HT/Step One etc.	5× (i.e.20μl system, add 2μl 50×ROX Reference Dye)
ABI 7500,7500 Fast, Stratagene Mx3000P, Mx3005P, and Mx4000, etc.	1× (i.e.20μl system, add 0.4μl 50×ROX Reference Dye)
Roche PCR instrument, Bio-Rad PCR instrument, Eppendorf quantitative PCR instrument, etc.	No need to add ROX Reference Dye

C:RT-qPCR reaction system setting

After preparing the RT-qPCR system with reference to the above table, mix gently (you can use a pipette tip to gently pipette; 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-qPCR reaction program settings (Table 2) to set the temperature and time of the reaction.

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

3. In order to get the best qPCR effect, gradient PCR can be used to optimize the reaction conditions for different templates and different primers.

Note: The extension temperature range of Foregene Hotstar Taq DNA Polymerase provided in this kit is: 60-72℃, and the best extension temperature is 72℃.

The following is an example of RT-qPCR reaction conditions. It is recommended to use a two-step method for PCR reaction. When the template concentration is too low to cause non-specific amplification, low primer Tm value leads to low amplification efficiency or poor amplification curve repeatability, etc., it is recommended to try the three-step method for PCR reaction. Refer to Table 2-1 (two-step method) and Table 2-2 (three-step method) for the setting of RT-qPCR reaction conditions.

Form 2-1:RT-qPCR reaction program setting(two step)

Step	Temperature	Time	Cycles	Content
1	42℃	10-30min 1*	1	Reverse Transcription
2	94-95℃	5min	1	Pre-denaturation
3	94-95℃	10sec	30-45	Template denaturation during cycles
3	60-65℃	20sec 2*	30-45	Annealing/Extension

Form 2-2:RT-qPCR reaction program setting( three step)

Step	Temperature	Time	Cycles	Content
1	42℃	10-30min 1*	1	Reverse Transcription
2	94-95℃	5min	1	Pre-denaturation
3	94-95℃	10sec	30-45	Template denaturation during cycles
	55-65℃	20sec		Primer annealing
	72℃	20-30sec 2*		Extension

1: Reverse transcription time can be adjusted according to experimental needs. General endogenous genes such as β-Actin only need 10 minutes; to detect specific expressed genes, reverse transcription time can be appropriately extended as needed.

2: Set a specific time according to the length of the amplified target fragment. The amplification speed of Foregene HotStar Taq DNA Polymerase is 2kb/min.

Note: PCR reaction conditions vary depending on the structural conditions of the template, primers, etc. For RNA templates with complex secondary structures, it is recommended to use 42℃ for the first step of reverse transcription. In the specific operation, it is necessary to design the optimal reaction conditions according to the specific conditions such as the size of the target fragment, the base sequence of the amplified fragment and the GC content and length of the primer, including annealing temperature, extension time, etc.

Work Flow

Precautions: (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.
The reagents in the kit should be protected from light and stored at -20°C.
It is recommended to use fresh sample extraction or template RNA stored at -80°C (RNA should avoid repeated freezing and thawing).
In order to avoid RNase contamination, please perform the experiment in the RNase-Free space; the pipette tips and PCR tubes used must be RNase-Free; and wear disposable gloves and masks.
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-qPCR EasyTM Mix-SYBR I 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 increase PCR amplification The specificity.
2×RT-qPCR EasyTM Mix-SYBR contains SYBR Green I, please avoid strong ligh

Documents

RT-qPCR Easy™ I (One Step)-SYBR Green I Instruction Manual-V1.0-EN

RT-qPCR EasyTM I (One Step)-SYBR Green I