基本描述

规格或纯度

High ROX

英文名称

UltraSYBR One Step RT-qPCR Kit

储存温度

避光,-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

产品内容

U665751	Component	100 T	Storage
U665751A	2×UltraSYBR One Step Buffer	1.4 mL	-20℃. Avoid freeze/ Thaw cycle. Protect from light.
U665751B	UltraSYBR One Step EnzymeMix	50 μL	-20℃. Avoid freeze/ Thaw cycle. Protect from light.
U665751C	50×High ROX	50 μL	-20℃. Avoid freeze/ Thaw cycle. Protect from light.
U665751D	RNase-Free Water	1.5 mL	-20℃. Avoid freeze/ Thaw cycle.

产品简介

本产品是一步法Real-Time RT-qPCR专用试剂盒。所含的SYBR Green I荧光染料可以与所有的双链DNA相结合，使该产品可以用于多种不同靶序列的检测而不需合成特异性标记探针。使用本产品进行Real Time RT-qPCR反应，逆转录和定量PCR在同一反应体系中进行，反应过程中无需添加试剂，无需打开管盖，避免了污染的同时提高了实验效率。全新高效逆转录酶RNase H活性缺失，减少了逆转录反应中RNA的降解。该酶逆转录效率高，可对少量 RNA模板进行良好的逆转录反应。与RNA亲合性高，能通读GC 含量高，二级结构复杂的RNA模板。全新高效热启动酶，在常温下酶的活性被封闭，从而有效避免在常温条件下由引物和模板非特异性结合或引物二聚体而产生的非特异性扩增，极大提高了荧光定量PCR反应的精确性。所包含的缓冲系统使以上两种酶同时发挥最大功效，提高效率。本产品灵敏度高，特异性高，线性范围广，对目的基因定量更准确。

ROX染料用于校正定量PCR仪孔与孔之间产生的荧光信号误差，一般用于ABI、 Stratagene等公司的Real Time PCR 扩增仪。不同仪器的激发光学系统有所不同，因此 ROX染料的浓度必须与相应的荧光定量PCR仪相匹配。

不需要ROX校正的仪器（U665567） :

Roche LightCycler 480，Roche LightCyler 96，Bio-rad iCyler iQ，iQ5，CFX96等。

需要High ROX校正的仪器（U665751）：

ABI Prism7000/7300/7700/7900，Eppendorf，ABI Step One/Step One Plus等。

注意事项

1．本试剂盒中的各试剂使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。

2．本产品以RNA为模板进行一步法RT-PCR实验，在操作过程中应避免RNase污染，建议在专门的区域进行RNA操作，使用专门的仪器和耗材，操作人员带口罩和一次性手套并经常更换手套，实验相关耗材应用0.1％DEPC(焦碳酸二乙酯)水溶液在37℃ 处理12小时,并高压灭菌30分钟后使用。注意事项试剂 25 μl反应体系终浓度 2×UltraSYBR One Step Buffer 12.5 μl 1× Forward Primer，10 µM 0.5 μl 0.2 μM1） Reverse Primer，10 µM 0.5 μl 0.2 μM1） 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

3．UltraSYBR One Step RT-qPCR Buffer中含有SYBR Green I 荧光染料，保存本产品或配制PCR反应液时应避免强光照射。

4．本试剂盒中的各试剂应避免反复冻融，反复冻融可能使产品性能下降。本产品长期保存可置于-20℃，避光。如果在短期内需要频繁使用，可在2-8℃保存。

5．本试剂盒必须使用特异性引物，引物的选择可根据具体实验来选择，引物设计的好坏直接影响到RT-PCR反应的结果，设计引物时需考虑GC含量，引物长度，引物位置，PCR产物的二级结构等因素，建议采用专业的引物设计软件进行设计。

6．本品不能用于探针法荧光定量PCR。

使用方法

1．将RNA模板、引物、2×UltraSYBR One Step Buffer、UltraSYBR One Step EnzymeMix和RNase-Free Water溶解并置于冰上备用。

2．PCR反应体系：

试剂	25 μl反应体系	终浓度
2×UltraSYBR One Step Buffer	12.5 μl	1×
Forward Primer，10 µM	0.5 μl	0.2 μM¹⁾
Reverse Primer，10 µM	0.5 μl	0.2 μM¹⁾
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

注意：1）通常引物浓度以0.2 µM可以得到较好结果，可以终浓度0.1-0.5 µM作为设定范围的参考。扩增效率不高的情况下，可提高引物的浓度；发生非特异性反应时，可降低引物浓度，由此优化反应体系。

2）不同仪器的激发光学系统有所不同，根据使用荧光定量的仪器选择加入50×Low ROX or 50×High ROX。

3．涡旋震荡混匀，短暂离心，将溶液收集到管底。

4．RT-qPCR反应条件(荧光定量PCR为两步法)，本程序是以ABI 7500荧光定量PCR 仪为例。

注意：1）建议采用两步法PCR反应程序，若提高反应特异性，可提高退火温度，以60-64℃作为设定范围的参考；若因使用Tm值较低的引物等原因，得不到良好的实验结果时，可尝试进行三步法PCR扩增。

2）融解曲线分析请以所使用的荧光定量PCR仪推荐的程序进行设定，本程序是以ABI 7500荧光定量PCR仪为参照设定。

RT-qPCR反应条件(荧光定量PCR为三步法)：

注意：1）三步法PCR扩增时，退火温度请以 56℃-64℃的范围作为设定参考。

2）融解曲线分析请以所使用的荧光定量PCR仪推荐的程序进行设定，本程序是以ABI 7500荧光定量PCR仪为参照设定。

Product content

U665751	Component	100 T	Storage
U665751A	2×UltraSYBR One Step Buffer	1.4 mL	-20℃. Avoid freeze/ Thaw cycle. Protect from light.
U665751B	UltraSYBR One Step EnzymeMix	50 μL	-20℃. Avoid freeze/ Thaw cycle. Protect from light.
U665751C	50×High ROX	50 μL	-20℃. Avoid freeze/ Thaw cycle. Protect from light.
U665751D	RNase-Free Water	1.5 mL	-20℃. Avoid freeze/ Thaw cycle.

Product Introduction

This product is a specialized kit for one-step Real-Time RT-qPCR. The SYBR Green I fluorescent dye contained can bind to all double-stranded DNA, allowing this product to be used for the detection of many different target sequences without the need to synthesize specific labeling probes. Real Time RT-qPCR reaction using this product, reverse transcription and quantitative PCR are carried out in the same reaction system, there is no need to add reagents during the reaction, no need to open the cap of the tube, avoiding contamination while improving the efficiency of the experiment. The new high-efficiency reverse transcriptase RNase H is activity-deficient, which reduces the degradation of RNA in the reverse transcription reaction. The enzyme has high reverse transcription efficiency and can perform a good reverse transcription reaction on a small amount of RNA template. It has high affinity to RNA and can read through RNA templates with high GC content and complex secondary structure. New efficient hot start enzyme, the enzyme activity is closed at room temperature, thus effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature, which greatly improves the accuracy of fluorescence quantitative PCR reaction. The included buffer system maximizes the efficacy of both enzymes at the same time and improves efficiency. This product has high sensitivity, high specificity, wide linear range, and more accurate quantification of target genes.

ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used with Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.

Instruments that do not require ROX calibration (U665567)

Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.

Instruments that require High ROX calibration (U665751)

ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.

matters needing attention

1. Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.

2. This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether of pyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes before use. Sterilize for 30 minutes before use.

3. UltraSYBR One Step RT-qPCR Buffer contains SYBR Green I fluorescent dye. Avoid bright light when storing this product or preparing PCR reaction solutions.

4. Repeated freezing and thawing of each reagent in this kit should be avoided; repeated freezing and thawing may degrade the product performance. This product can be stored for a long time at -20℃, protected from light. If frequent use is required in the short term, it can be stored at 2-8℃.

5. This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-PCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.

6. This product cannot be used for fluorescent quantitative PCR by the probe method.

Usage

1. Dissolve RNA template, primers, 2× UltraSYBR One Step Buffer, UltraSYBR One Step EnzymeMix and RNase-Free Water and set aside on ice.

2. PCR reaction system:

Reagents	25 μl Reaction system	Final concentration
2×UltraSYBR One Step Buffer	12.5 μl	1×
Forward Primer，10 µM	0.5 μl	0.2 μM¹⁾
Reverse Primer，10 µM	0.5 μl	0.2 μM¹⁾
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

Note: 1) Usually, the primer concentration of 0.2µM can get better results, and the final concentration of 0.1-0.5µM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; when non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.

(2) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.

3. Vortex and shake to mix, centrifuge briefly, and collect the solution at the bottom of the tube.

4. RT-qPCR reaction conditions (fluorescence quantitative PCR is a two-step method), this program is based on the ABI 7500 fluorescence quantitative PCR instrument as an example

Note: 1) It is recommended to use two-step PCR reaction program, if you improve the reaction specificity, you can increase the annealing temperature to 60-64 ℃ as a reference for the setting range; if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification.

(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument used, and this program is set up with the ABI 7500 fluorescence quantitative PCR instrument as a reference.

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

Note: 1) For three-step PCR amplification, please use the range of 56℃-64℃ as the setting reference for the annealing temperature.

(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument you are using, this program is ABI

750 fluorescent quantitative PCR instrument as a reference setting.

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