第一链cDNA合成试剂盒|Hifair® Ⅱ 1st Strand cDNA Synthesis Kit

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第一链cDNA合成试剂盒|Hifair® Ⅱ 1st Strand cDNA Synthesis Kit

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产品描述

Hifair® Ⅱ 1st Strand cDNA Synthesis Kit是基于Hifair® Ⅱ Reverse Transcriptase开发的试剂盒。与Hieff® M-MLV (H–) Reverse Transcriptase相比，Hifair® Ⅱ Reverse Transcriptase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。Hifair® Ⅱ Reverse Transcriptase合成全长cDNA的能力也有了提升，可扩增长达10 kb的cDNA。
试剂盒中包含由总RNA或mRNA合成高质量第一链cDNA所需的所有成分，并提供两种cDNA合成引物：Random Primers N6和oligo (dT)18，合成的单链cDNA产物可直接用来进行后续PCR或者qPCR反应。

产品组分
 

编号

组分

产品编号/规格

11119ES60 (100 T)

11119-A

RNase-free H2O

2×1 mL

11119-B

5×Hifair® Ⅱ Buffer

400 μL

11119-C

Hifair® Ⅱ Enzyme Mix

200 μL

11119-D

Oligo (dT)18 (50 μM)

100 μL

11119-E

Random Primers N6 (50 μM)

100 μL

【注】：1）5×Hifair® Ⅱ Buffer中包含dNTP。2）Hifair® Ⅱ Enzyme Mix中包含RNase inhibitor。

运输和保存方法

冰袋运输。-20℃保存。

注意事项

1）反应液的配制应在冰上操作完成，操作过程应避免RNase污染；

2)  为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
3）本产品仅作科研用途！

 

第一链cDNA合成操作步骤
 

1. 逆转录反应体系配制（20 μL体系）

组分

使用量

RNase-free H2O

To 20 μL

Total RNA

1 ng -5 μg* 

or mRNA

1 ng-500 ng*

5×Hifair® Ⅱ Buffer

4 μL

Random Primers N6 (50 μM)

1 μL

or Oligo (dT)18 (50 μM)

or 1 μL

or Gene Specific Primers (2 μM)

or 1 μL

Hifair® Ⅱ Enzyme Mix

2 μL

【可省略】：若后续实验为PCR，针对复杂模板，可将RNA、H2O、反转录引物在65℃孵育5 min 后，迅速冰上冷却，然后再加入Hifair® Ⅱ Enzyme Mix；若后续实验为qPCR，则可省略65℃孵育步骤，直接在体系中加入Hifair® Ⅱ Enzyme Mix。

【注】：* 若后续实验为qPCR，建议Total RNA 或 mRNA投入量不超过1 μg或100 ng，如果目的基因的表达丰度非常低，最多可投入5 μg Total RNA或500 ng mRNA。

2. 逆转录程序设置

温度

时间

25℃

5 min

42℃

30 min

85℃

5 min

【注】：
1）荧光定量实验可以只使用Random Primers N6；也可以1:1与Oligo (dT)18混合使用，效果更好。
2）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℃。
3）逆转录产物可以-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。

关于引物的选择
 

1） 如果模板为真核生物来源，建议选择Oligo (dT)18 ，与真核生物mRNA的3’ Poly A尾配对，可获得最高产量的全长cDNA。
2）原核生物RNA的反转录请选用Random Primers N6或者基因特异性引物。

3）Random Primers N6适用性较广，mRNA、rRNA、tRNA、small RNA和LncRNA等模板均可用Random Primers N6进行反转录。

4）使用Random primers N6，进行2 kb以下的cDNA合成时，Random primers N6的使用量为 1-2 μL；2 kb 以上的cDNA合成时，Random primers N6 的使用量为0.4-1 μL。

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

11123ES60

100 T

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

11139ES60

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

HB210719

Q：A2800，promage 的货号， 用我们哪款反转录替换比较好点？

A：11119 是两种引物分开的，可以做 oligo（dT）和随机引物分开的，也可以混合的。如果客户只做定量的话，可以推 11137

Q：问一下 11110 这款反转录单酶，这款产品里头的组分有没有那款kit 或者 mix 也包含呢？

A：11120ES、11123ES、11119ES、11121ES

Q：Hifair Ⅱ 1st Strand cDNA Synthesis Kit 这个跟诺唯赞的 HiScript II Reverse Transcriptase 区别？

A：如果客户不要求逆转温度达到 55 度，是可以的，我们三代逆转录酶优于这个。

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产品描述

Hifair® Ⅱ 1st Strand cDNA Synthesis Kit是基于Hifair® Ⅱ Reverse Transcriptase开发的试剂盒。与Hieff® M-MLV (H–) Reverse Transcriptase相比，Hifair® Ⅱ Reverse Transcriptase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。Hifair® Ⅱ Reverse Transcriptase合成全长cDNA的能力也有了提升，可扩增长达10 kb的cDNA。
试剂盒中包含由总RNA或mRNA合成高质量第一链cDNA所需的所有成分，并提供两种cDNA合成引物：Random Primers N6和oligo (dT)18，合成的单链cDNA产物可直接用来进行后续PCR或者qPCR反应。

产品组分
 

编号

组分

产品编号/规格

11119ES60 (100 T)

11119-A

RNase-free H2O

2×1 mL

11119-B

5×Hifair® Ⅱ Buffer

400 μL

11119-C

Hifair® Ⅱ Enzyme Mix

200 μL

11119-D

Oligo (dT)18 (50 μM)

100 μL

11119-E

Random Primers N6 (50 μM)

100 μL

【注】：1）5×Hifair® Ⅱ Buffer中包含dNTP。2）Hifair® Ⅱ Enzyme Mix中包含RNase inhibitor。

运输和保存方法

冰袋运输。-20℃保存。

注意事项

1）反应液的配制应在冰上操作完成，操作过程应避免RNase污染；

2)  为了您的安全和健康，请穿实验服并佩戴一次性手套操作。
3）本产品仅作科研用途！

 

第一链cDNA合成操作步骤
 

1. 逆转录反应体系配制（20 μL体系）

组分

使用量

RNase-free H2O

To 20 μL

Total RNA

1 ng -5 μg* 

or mRNA

1 ng-500 ng*

5×Hifair® Ⅱ Buffer

4 μL

Random Primers N6 (50 μM)

1 μL

or Oligo (dT)18 (50 μM)

or 1 μL

or Gene Specific Primers (2 μM)

or 1 μL

Hifair® Ⅱ Enzyme Mix

2 μL

【可省略】：若后续实验为PCR，针对复杂模板，可将RNA、H2O、反转录引物在65℃孵育5 min 后，迅速冰上冷却，然后再加入Hifair® Ⅱ Enzyme Mix；若后续实验为qPCR，则可省略65℃孵育步骤，直接在体系中加入Hifair® Ⅱ Enzyme Mix。

【注】：* 若后续实验为qPCR，建议Total RNA 或 mRNA投入量不超过1 μg或100 ng，如果目的基因的表达丰度非常低，最多可投入5 μg Total RNA或500 ng mRNA。

2. 逆转录程序设置

温度

时间

25℃

5 min

42℃

30 min

85℃

5 min

【注】：
1）荧光定量实验可以只使用Random Primers N6；也可以1:1与Oligo (dT)18混合使用，效果更好。
2）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℃。
3）逆转录产物可以-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。

关于引物的选择
 

1） 如果模板为真核生物来源，建议选择Oligo (dT)18 ，与真核生物mRNA的3’ Poly A尾配对，可获得最高产量的全长cDNA。
2）原核生物RNA的反转录请选用Random Primers N6或者基因特异性引物。

3）Random Primers N6适用性较广，mRNA、rRNA、tRNA、small RNA和LncRNA等模板均可用Random Primers N6进行反转录。

4）使用Random primers N6，进行2 kb以下的cDNA合成时，Random primers N6的使用量为 1-2 μL；2 kb 以上的cDNA合成时，Random primers N6 的使用量为0.4-1 μL。

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

11123ES60

100 T

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

11139ES60

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

HB210719

Q：A2800，promage 的货号， 用我们哪款反转录替换比较好点？

A：11119 是两种引物分开的，可以做 oligo（dT）和随机引物分开的，也可以混合的。如果客户只做定量的话，可以推 11137

Q：问一下 11110 这款反转录单酶，这款产品里头的组分有没有那款kit 或者 mix 也包含呢？

A：11120ES、11123ES、11119ES、11121ES

Q：Hifair Ⅱ 1st Strand cDNA Synthesis Kit 这个跟诺唯赞的 HiScript II Reverse Transcriptase 区别？

A：如果客户不要求逆转温度达到 55 度，是可以的，我们三代逆转录酶优于这个。

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