Fast Ampli RT-qPCR Premix plus-UNG

FastAmpli RT-qPCR Premix-UNG (Lyophilized powder) is a pre-packed lyophilized reagent designed for one-step fluorescent quantitative PCR using probes. This product contains high-temperature reverse transcriptase with RNase H activity removed and DNA polymerase that have been genetically modified to achieve rapid amplification.

Cat No: HYA432

Package: 100RXN/1000RXN/10000RXN

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Product Description

Product detail

Product Tags

Cat No: HCB5144E

Fast Ampli RT-qPCR Premix plus-UNG is developed specifically for lyophilization processes, applied for fluorescence quantification (TaqMan probe) RNA amplification detection containing neocript reverse transcriptase and rapid amplification DNA Polymerase obtained by genetically modified and screening which can complete the PCR amplification within 20-40 minutes. This reagent preforms high inhibitor- tolerance, which has higher reverse transcription and PCR amplification efficiency, suitable for high-sensitivity amplification of low-concentration RNA samples. A good standard curve can be obtained within a wide quantitative range, and quantification can be carried out accurately. This reagent uses a mixed enzyme of anti-inhibition amplification enzyme and UNG enzyme, as well as an optimized buffer system containing dUTP. It can not only achieve good amplification of the target gene in samples containing inhibitors, but also effectively prevent false positive amplification caused by PCR residual and aerosol pollution. This reagent is compatible with most fluorescent quantitative PCR instruments, such as Applied Biosystems, Eppendorf, Bio-Rad, Roche and so on. This reagent can be used for lyophilization to obtain a good lyophilized form and product stability.

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Product Components

FastAmpli RT-qPCR Premix-UNG (Lyophilized powder) contains rapid Taq DNA polymerase,Reverse Transcriptase, PCR Buffer, UNG, dNTPs (with dUTP instead of dTTP), MgCl2, stabilizer and lyoprotectants.

Storage Conditions

The product store at 4℃ or room temperature.

Instructions

1. Reaction System

Composition	Volume (20 μL reaction)
FastAmpli RT-qPCR Premix-UNG (Lyophilized powder)	4μL
25X Primer Probe Mix 1,2	1μl
Template RNA3	X μL
ddH2O	up to 25μL

Notes: Reaction volume is 10-50μL.

1. Typically 0.2μM primer concentration yields better results; if reaction performance is poor then adjust primerconcentration between 0.2~1μ M range accordingly. Typically probe concentrations are optimized within

therange of 0.1~0.3μ M. Concentration gradient experiments may be performed to find the best combination of primers and probes.

2. When using a fast PCR procedure, increasing the concentration of primers and probes may result in betteramplification results, and their ratio should be optimized accordingly

3. The copy number of target genes contained in different types of templates differs; therefore, it may benecessary to perform gradient dilution experiments to determine the optimal amount of template addition.

2.Cycle Protocol(Standard)

Cycle step	Temp.	Time	Cycles
Reverse Transcription	50℃	10-20 min	1
Initial Denaturation	95℃	1-5 min	1
Denaturation	95℃	10-20 s	40-45
Annealing/Extension	56-64℃	20-60 s	40-45

3.Cycle Protocol(Fast)

Cycle step	Temp.	Time	Cycles
Reverse Transcription	50℃	5 min	1
Initial Denaturation	95℃	30 s	1
Denaturation	95℃	1-3 s	40-45
Annealing/Extension	56-64℃	3-20 s	40-45

*The temperature-sensitive UNG enzyme contained in this product can function at room temperature and become

inactive during the reverse transcription process.

Technical Information

1. Amplification rate of rapid DNA polymerase is no less than 1kb/10s. Different PCR instruments have different heating and cooling speeds, temperature control modes and thermal conductivity, and thus

optimization of your primer/probe concentration and running method in combination with your specific fast PCR instrument is essential.

2. Reverse transcription time should be optimized according to the length of the fragment being amplified; longer fragments may require a longer reverse transcription time.

3. Annealing extension temperature should be optimized based on primer probe TM values and actual reaction conditions.

4. The three-step PCR method is recommended for primers with low annealing temperature or for amplification of long fragments over 200 bp.

5. Since different amplicons have different utilization efficiency to dUTP and different sensitivity to UNG, the reagents should be optimized if the detection sensitivity decreases when using UNG system. Please contact us for technical support if needed.

6. To avoid amplification of carryover PCR products, dedicated experimental area and pipette are required for amplification. Operate with gloves and change frequently and do not open the PCR tube after amplification.

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

Guidelines for lyophilized products

1. Divide the lyophilized sphere into PCR tube.

2. Add Primer-Probe Mix and templates to the lyophilized powder, and add water up to 25μL.

3. . Mix and centrifuge, and then proceed with PCR amplification