UltraSYBR One Step RT-qPCR Kit - 100 rxns, high purity

Item Number

U665567

• Storage Temp: Protected from light,Store at -20°C,Avoid repeated freezing and thawing
• Shipped In: Ice chest + Ice pads

Product Description

U665567	Component	100 T	Storage
U665567A	2×UltraSYBR One Step Buffer	1.4 mL	-20℃. Avoid freeze/thaw cycle. Protect from light.
U665567B	UltraSYBR One Step EnzymeMix	50 μL	-20℃. Avoid freeze/thaw cycle. Protect from light.
U665567C	RNase-Free Water	1.5 mL	-20℃. Avoid freeze/thaw cycle.

This product is a one-step Real Time RT qPCR specialized reagent kit. The SYBR Green I fluorescent dye contained can bind to all double stranded DNA, making the product suitable for detecting various target sequences without the need for synthesizing specific labeled probes. Using this product for Real Time RT qPCR reaction, reverse transcription and quantitative PCR are performed in the same reaction system, without the need to add reagents or open the tube cap during the reaction process, avoiding contamination and improving experimental efficiency. The loss of RNase H activity, a novel and efficient reverse transcriptase, reduces the degradation of RNA in reverse transcription reactions. This enzyme has high reverse transcription efficiency and can perform good reverse transcription reactions on a small amount of RNA templates. High affinity with RNA, able to read RNA templates with high GC content and complex secondary structures. A new and efficient hot start enzyme, whose activity is blocked at room temperature, effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimers at room temperature, greatly improving the accuracy of fluorescence quantitative PCR reactions. The included buffer system maximizes the effectiveness of both enzymes and improves efficiency. This product has high sensitivity, specificity, wide linear range, and more accurate quantification of target genes. ROX dye is used to correct the fluorescence signal error between wells in quantitative PCR instruments, and is generally used in Real Time PCR amplification instruments from companies such as ABI and Stratagene. The excitation optical systems of different instruments vary, so the concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument. 

Instruments that do not require ROX calibration( U665567 ): Roche LightCycle 480, Roche LightCyler 96, Bio rad iCyler iQ, iQ5, CFX96, etc. 

Instruments that require High ROX calibration include( U665751）：ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.

Component	U665567 100 rxns
2×UltraSYBR One Step Buffer	1.4 mL
UltraSYBR One Step EnzymeMix	50 μL
50×Low ROX	/
50×High ROX	/
RNase-Free Water	1.5 mL

Notes:
1. Before use, please gently mix the reagents in this reagent kit upside down to avoid foaming, and use after brief centrifugation.
2. This product uses RNA as a template for one-step RT-PCR experiments. During the operation, RNase contamination should be avoided. It is recommended to perform RNA operations in a dedicated area, using specialized instruments and consumables. Operators should wear masks and disposable gloves and frequently change gloves. The experimental consumables should be treated with 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours and sterilized under high pressure for 30 minutes before use.
3. UltraSYBR One Step RT qPCR Buffer contains SYBR Green I fluorescent dye. When storing this product or preparing PCR reaction solution, avoid strong light irradiation.
4. Each reagent in this kit should avoid repeated freeze-thaw cycles, as repeated freeze-thaw cycles may lead to a decrease in product performance. This product can be stored for a long time at -20 ℃, away from light. If frequent use is required in the short term, it can be stored at 2-8 ℃.
5. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT-PCR reactions. When designing primers, factors such as GC content, primer length, primer position, and the secondary structure of PCR products need to be considered. It is recommended to use professional primer design software for design.
6. This product cannot be used for probe based fluorescence quantitative PCR.

Usage:
1. Dissolve the RNA template, primers, 2x UltraSYBR One Step Buffer, UltraSYBR One Step EnzymeMix, and RNase Free Water and place them on ice for later use.
2. PCR reaction system:

Reagent	25 μL Reaction system	Final concentration
2×UltraSYBR One Step Buffer	12.5 μL	1×
Forward Primer，10 µM	0.5 μL	0.2 μM ¹⁾
Reverse Primer，10 µM	0.5 μL	0.2 μM ¹⁾
UltraSYBR One Step EnzymeMix	0.5 μL	/
RNA Template	X μL	10 pg – 100 ng
50×Low ROX or High ROX（optional）²⁾	0.5 μL	1×
RNase-Free Water	up to 25 μL	/

Attention:
1) Typically, the primer concentration is 0.2 μ M can obtain good results, with a final concentration of 0.1-0.5 μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.
2) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added.

3. Vortex and shake well, centrifuge briefly, and collect the solution to the bottom of the tube.
4. RT qPCR reaction conditions (fluorescence quantitative PCR is a two-step method). This program takes the ABI 7500 fluorescence quantitative PCR instrument as an example.

Step	Temperature	Time	/
Reverse transcription	45℃	10 min	/
PCR pre denaturation	95℃	5 min	/
Denaturation	95℃	10 s	30-40 cycles
Annealing/Extension ¹ ⁾	60℃	45 s	30-40 cycles
Analysis of Fusion Curve ² ⁾	/	/	/
/	95℃	15 s	/
/	60℃	1 min	/
/	95℃	15 s	/
/	60℃	15 s	/

RT qPCR reaction conditions (fluorescence quantitative PCR is a three-step method):

Step	Temperature	Time	/
Reverse transcription	45℃	10 min	/
PCR pre denaturation	95℃	5 min	/
Denaturation	95℃	10 s	35-40 cycles
Annealing ¹ ⁾	56℃-64℃	30 s	35-40 cycles
Extension	72℃	30 s	35-40 cycles
Analysis of Fusion Curve ² ⁾	/	/	/
/	95℃	15 s	/
/	60℃	1 min	/
/	95℃	15 s	/
/	60℃	15 s	/

Attention:
1) When using the three-step PCR amplification method, the annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference.
2) Please set the fusion curve analysis according to the recommended program of the fluorescence quantitative PCR instrument used. This program is based on the ABI 7500 fluorescence quantitative PCR instrument as a reference setting.