qPCR第一链cDNA合成即用型预混液(含有gDNA去除)|1st Strand cDNA Synthesis SuperMix for qPCR(gDNA digester plus)

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qPCR第一链cDNA合成即用型预混液(含有gDNA去除)|1st Strand cDNA Synthesis SuperMix for qPCR(gDNA digester plus)

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产品描述

 

Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)基于Hifair® Ⅱ Reverse Transcriptase而开发的即用型预混液。该酶的热稳定性大幅度提高，可耐受高达50℃的反应温度，适合具有复杂二级结构的RNA模板的逆转录。同时，该酶增强了与模板的亲和力，适合少量模板以及低拷贝基因的逆转录。

该预混液包含gDNA digester和2×Hifair® Ⅱ SuperMix plus。gDNA digester可去除RNA模板中残留的基因组DNA污染，保证后续结果更加可靠。2×Hifair® Ⅱ SuperMix plus含有逆转录反应所需的所有组分（Buffer，dNTP，Hifair® Ⅱ Reverse Transcriptase，RNase inhibitor，Random primers/ Oligo (dT)18 primer mix），只需加入RNA模板和 RNase-free ddH2O即可进行逆转录反应，并同时终止gDNA digester的作用，保证cDNA的完整性。

该产品适用于两步法RT-qPCR检测，针对qPCR进行Random primers/ Oligo (dT)18 primer的比例优化，使cDNA合成可从RNA转录本的各个区域起始，并具有相同的逆转录效率，最大程度保证了qPCR结果的真实性和可重复性。逆转录产物兼容SYBR® Green和探针法qPCR，可以根据实验目的，选择UNICON® qPCR SYBR® Green Master Mix，Hieff® qPCR SYBR® Green Master Mix或Hieff® qPCR TaqMan Probe Master Mix等试剂配合使用，进行高性能的基因表达分析。

 

产品组分

 

编号

组分

产品编号/规格

11123ES10 (10 T)

11123ES60 (100 T)

11123-A

RNase-free ddH2O

1 mL

2×1 mL

11123-B

5×gDNA digester Buffer

20 μL

200 μL

11123-C

gDNA digester

10 μL

100 μL

11123-D

2×Hifair® Ⅱ SuperMix plus

100 μL

1 mL

【注】：2×Hifair® Ⅱ SuperMix plus含有gDNA digester抑制剂。

 

运输和保存方法

干冰运输。-20℃保存。有效期18个月。

 

注意事项

1）gDNA digester和2×Hifair® Ⅱ SuperMix plus含有高浓度的甘油，使用前请短暂离心，吹打混匀。

2）建议RNA是溶于水而不是TE Buffer中，因为TE Buffe会干扰gDNA去除以及逆转录反应。

3）可以不经过基因组去除步骤，直接进行逆转录，这样所得到的结果与使用Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix  (Cat No. 11120ES) 效果一致。但是请勿将gDNA digester与11120ES中的2×Hifair® Ⅱ SuperMix配套使用，因其不含终止gDNA digester反应的成分，会影响反转录和后续的qPCR实验。

4）反应液的配制应在冰上操作完成，操作过程应避免RNase污染。

5) 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。

6) 本产品仅作科研用途！

 

第一链cDNA合成操作步骤

 

1. 残留基因组DNA去除

在RNase free离心管中配制如下混合液，用移液器轻轻吹打混匀。42℃ 孵育2 min。

组分

使用量

RNase free ddH2O

To 10 μL

5×gDNA digester Buffer

2 μL

gDNA digester

1 μL

Total RNA

1 ng -5 μg*

or mRNA

1 ng-500 ng*

【注】：* 20 μL逆转录反应体系建议Total RNA的投入量不超过1 μg。如果目的基因的表达丰度低，最多投入5 μg Total RNA，否则RNA投入量过高，可能会超过后续定量PCR的线性范围。

2. 逆转录反应体系配制（20 μL体系）

在第1步的反应管中直接加入2×HifairTM Ⅱ SuperMix plus，用移液器轻轻吹打混匀。

组分

使用量

第1步的反应液

10 μL

2×Hifair® Ⅱ SuperMix plus

10 μL

3. 逆转录程序设置

标准程序（适用于微量和常规量的模板量）

温度

时间

25℃

5 min

42℃

30 min

85℃

5 min

【注】：逆转录温度：推荐使用42℃。对于高GC含量模板或者复杂模板，可将逆转录温度提高到50℃。

4. 逆转录产物可立即用于qPCR反应，也可-20℃短期保存，若需长期保存，建议分装后，于-80℃保存，避免反复冻融。

 

相关产品

 

产品名称

货号

规格

Hifair® II 1st Strand cDNA Synthesis Kit 

11119ES60

100 T

Hifair® II 1st Strand cDNA Synthesis SuperMix

11120ES60

100 T

Hifair® II 1st Strand cDNA Synthesis Kit (gDNA digester plus)

11121ES60

100 T

Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)

11141ES60

100 T

Hifair® qPCR SYBR® Green Master Mix (No Rox )

11201ES08

5 mL

Hifair® qPCR SYBR® Green Master Mix (Low Rox Plus) 

11202ES08

5 mL

Hifair® qPCR SYBR® Green Master Mix (High Rox Plus) 

11203ES08

5 mL

Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，No Rox)

11195ES08

5 mL

Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，Low Rox)

11196ES08

5 mL

Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，High Rox)

11197ES08

5 mL

Hieff UNICON® qPCR SYBR Green Master Mix ( 抗体法，No Rox)

11198ES08

5 mL

Hieff UNICON® qPCR SYBR Green Master Mix ( 抗体法，Low Rox)

11199ES08

5 mL

Hieff UNICON® qPCR SYBR Green Master Mix ( 抗体法，High Rox)

11200ES08

5 mL

Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix

11184ES08

5 mL

                                                                                              HB210628

 

 

 

Q：逆转录产品如何选择？

 

 

A：参考下表。

产品名称/货号

产品形式

引物

适用实验类型

产品特点/适用范围（A/B）

Hifair™ II Reverse Transcriptase

第二代耐热逆转录酶  11110ES

单酶

无

☑cDNA产物后续用于PCR

☑cDNA产物后续用于qPCR

A．常规表达丰度RNA逆转录（总RNA   1ng-5μg）

Hifair™ III Reverse Transcriptase

第三代耐热逆转录酶  11111ES

B.低表达丰度RNA逆转录

（总RNA 10pg-5μg）；复杂RNA

Hifair™ II 1st Strand cDNA Synthesis Kit   11119ES/11121ES

酶和Buffer分开

单独成管的Oligo dT 、Random primer

同A

Hifair™ III 1st Strand cDNA Synthesis Kit 11135ES/11139ES

同B

Hifair™ II 1st Strand cDNA Synthesis SuperMix for qPCR  11120ES/11123ES

酶和Buffer预混

按比例预混的Oligo dT 、Random primer

☑cDNA产物后续用于qPCR

同A

Hifair™ III 1st Strand cDNA Synthesis SuperMix for qPCR  11137ES/11141ES

同B

Q：逆转录引物如何选择？
 

A：11123ES中使用的是Random Primers和oligo dT按照一定优化的比例预混引物。
 

Q：反转录产物可以做普通PCR吗？
 

A：不推荐。11123ES以预混液形式提供，预混液中包含Oligo (dT)与Random Primers混合引物，逆转录产物反转均一，但长度较短，可以做短片段，过长的不适合。若反转录产物后续进行PCR，建议用翌圣11121ES，引物选oligo dT。
 

Q：逆转录产物后续进行qPCR，需要稀释吗？

A：建议稀释5-10倍。目的有二：一、提高稀释倍数，降低移液误差，提高实验重复性；二、降低逆转录体系中高浓度某些成分对下游qPCR反应的抑制作用。
 

Q：该产品可以搭配其他品牌的定量试剂使用吗？
 

A：可以。逆转录产物进行稀释后，反转录相关组分对后续不同品牌的qPCR试剂影响非常小。

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产品描述

 

Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)基于Hifair® Ⅱ Reverse Transcriptase而开发的即用型预混液。该酶的热稳定性大幅度提高，可耐受高达50℃的反应温度，适合具有复杂二级结构的RNA模板的逆转录。同时，该酶增强了与模板的亲和力，适合少量模板以及低拷贝基因的逆转录。

该预混液包含gDNA digester和2×Hifair® Ⅱ SuperMix plus。gDNA digester可去除RNA模板中残留的基因组DNA污染，保证后续结果更加可靠。2×Hifair® Ⅱ SuperMix plus含有逆转录反应所需的所有组分（Buffer，dNTP，Hifair® Ⅱ Reverse Transcriptase，RNase inhibitor，Random primers/ Oligo (dT)18 primer mix），只需加入RNA模板和 RNase-free ddH2O即可进行逆转录反应，并同时终止gDNA digester的作用，保证cDNA的完整性。

该产品适用于两步法RT-qPCR检测，针对qPCR进行Random primers/ Oligo (dT)18 primer的比例优化，使cDNA合成可从RNA转录本的各个区域起始，并具有相同的逆转录效率，最大程度保证了qPCR结果的真实性和可重复性。逆转录产物兼容SYBR® Green和探针法qPCR，可以根据实验目的，选择UNICON® qPCR SYBR® Green Master Mix，Hieff® qPCR SYBR® Green Master Mix或Hieff® qPCR TaqMan Probe Master Mix等试剂配合使用，进行高性能的基因表达分析。

 

产品组分

 

编号

组分

产品编号/规格

11123ES10 (10 T)

11123ES60 (100 T)

11123-A

RNase-free ddH2O

1 mL

2×1 mL

11123-B

5×gDNA digester Buffer

20 μL

200 μL

11123-C

gDNA digester

10 μL

100 μL

11123-D

2×Hifair® Ⅱ SuperMix plus

100 μL

1 mL

【注】：2×Hifair® Ⅱ SuperMix plus含有gDNA digester抑制剂。

 

运输和保存方法

干冰运输。-20℃保存。有效期18个月。

 

注意事项

1）gDNA digester和2×Hifair® Ⅱ SuperMix plus含有高浓度的甘油，使用前请短暂离心，吹打混匀。

2）建议RNA是溶于水而不是TE Buffer中，因为TE Buffe会干扰gDNA去除以及逆转录反应。

3）可以不经过基因组去除步骤，直接进行逆转录，这样所得到的结果与使用Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix  (Cat No. 11120ES) 效果一致。但是请勿将gDNA digester与11120ES中的2×Hifair® Ⅱ SuperMix配套使用，因其不含终止gDNA digester反应的成分，会影响反转录和后续的qPCR实验。

4）反应液的配制应在冰上操作完成，操作过程应避免RNase污染。

5) 为了您的安全和健康，请穿实验服并佩戴一次性手套操作。

6) 本产品仅作科研用途！

 

第一链cDNA合成操作步骤

 

1. 残留基因组DNA去除

在RNase free离心管中配制如下混合液，用移液器轻轻吹打混匀。42℃ 孵育2 min。

组分

使用量

RNase free ddH2O

To 10 μL

5×gDNA digester Buffer

2 μL

gDNA digester

1 μL

Total RNA

1 ng -5 μg*

or mRNA

1 ng-500 ng*

【注】：* 20 μL逆转录反应体系建议Total RNA的投入量不超过1 μg。如果目的基因的表达丰度低，最多投入5 μg Total RNA，否则RNA投入量过高，可能会超过后续定量PCR的线性范围。

2. 逆转录反应体系配制（20 μL体系）

在第1步的反应管中直接加入2×HifairTM Ⅱ SuperMix plus，用移液器轻轻吹打混匀。

组分

使用量

第1步的反应液

10 μL

2×Hifair® Ⅱ SuperMix plus

10 μL

3. 逆转录程序设置

标准程序（适用于微量和常规量的模板量）

温度

时间

25℃

5 min

42℃

30 min

85℃

5 min

【注】：逆转录温度：推荐使用42℃。对于高GC含量模板或者复杂模板，可将逆转录温度提高到50℃。

4. 逆转录产物可立即用于qPCR反应，也可-20℃短期保存，若需长期保存，建议分装后，于-80℃保存，避免反复冻融。

 

相关产品

 

产品名称

货号

规格

Hifair® II 1st Strand cDNA Synthesis Kit 

11119ES60

100 T

Hifair® II 1st Strand cDNA Synthesis SuperMix

11120ES60

100 T

Hifair® II 1st Strand cDNA Synthesis Kit (gDNA digester plus)

11121ES60

100 T

Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)

11141ES60

100 T

Hifair® qPCR SYBR® Green Master Mix (No Rox )

11201ES08

5 mL

Hifair® qPCR SYBR® Green Master Mix (Low Rox Plus) 

11202ES08

5 mL

Hifair® qPCR SYBR® Green Master Mix (High Rox Plus) 

11203ES08

5 mL

Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，No Rox)

11195ES08

5 mL

Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，Low Rox)

11196ES08

5 mL

Hieff UNICON® Power qPCR SYBR Green Master Mix ( 抗体法，High Rox)

11197ES08

5 mL

Hieff UNICON® qPCR SYBR Green Master Mix ( 抗体法，No Rox)

11198ES08

5 mL

Hieff UNICON® qPCR SYBR Green Master Mix ( 抗体法，Low Rox)

11199ES08

5 mL

Hieff UNICON® qPCR SYBR Green Master Mix ( 抗体法，High Rox)

11200ES08

5 mL

Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix

11184ES08

5 mL

                                                                                              HB210628

 

 

 

Q：逆转录产品如何选择？

 

 

A：参考下表。

产品名称/货号

产品形式

引物

适用实验类型

产品特点/适用范围（A/B）

Hifair™ II Reverse Transcriptase

第二代耐热逆转录酶  11110ES

单酶

无

☑cDNA产物后续用于PCR

☑cDNA产物后续用于qPCR

A．常规表达丰度RNA逆转录（总RNA   1ng-5μg）

Hifair™ III Reverse Transcriptase

第三代耐热逆转录酶  11111ES

B.低表达丰度RNA逆转录

（总RNA 10pg-5μg）；复杂RNA

Hifair™ II 1st Strand cDNA Synthesis Kit   11119ES/11121ES

酶和Buffer分开

单独成管的Oligo dT 、Random primer

同A

Hifair™ III 1st Strand cDNA Synthesis Kit 11135ES/11139ES

同B

Hifair™ II 1st Strand cDNA Synthesis SuperMix for qPCR  11120ES/11123ES

酶和Buffer预混

按比例预混的Oligo dT 、Random primer

☑cDNA产物后续用于qPCR

同A

Hifair™ III 1st Strand cDNA Synthesis SuperMix for qPCR  11137ES/11141ES

同B

Q：逆转录引物如何选择？
 

A：11123ES中使用的是Random Primers和oligo dT按照一定优化的比例预混引物。
 

Q：反转录产物可以做普通PCR吗？
 

A：不推荐。11123ES以预混液形式提供，预混液中包含Oligo (dT)与Random Primers混合引物，逆转录产物反转均一，但长度较短，可以做短片段，过长的不适合。若反转录产物后续进行PCR，建议用翌圣11121ES，引物选oligo dT。
 

Q：逆转录产物后续进行qPCR，需要稀释吗？

A：建议稀释5-10倍。目的有二：一、提高稀释倍数，降低移液误差，提高实验重复性；二、降低逆转录体系中高浓度某些成分对下游qPCR反应的抑制作用。
 

Q：该产品可以搭配其他品牌的定量试剂使用吗？
 

A：可以。逆转录产物进行稀释后，反转录相关组分对后续不同品牌的qPCR试剂影响非常小。

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