RevertAid First Strand cDNA Synthesis Kit

Features

·         Full-length first strand cDNA up to 13 kb.

·         Increased reaction temperatures in the range of 42-50°C.

·         Supplied with the recombinant RiboLock™ RNase Inhibitor.

·         Complete – oligo(dT)₁₈ and random hexamer primers included with the kit.

Applications

·         First strand cDNA synthesis for RT-PCR and real-time RT-PCR (1, 2).

·         Construction of full length cDNA libraries.

·         Generation of probes for hybridization.

·         aRNA synthesis.

Description

The RevertAid™ First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit is suitable for synthesis of cDNA up to 13 kb.

The kit uses RevertAid™ Reverse Transcriptase which has lower RNase H activity, compared to AMV reverse transcriptase.

The recombinant RiboLock™ RNase Inhibitor, supplied with the kit, effectively protects RNA template from degradation. It is fully compatible with reverse transcription reaction, as it maintains activity at temperatures up to 55°C.

The kit is supplied with both oligo(dT)₁₈ and random hexamer primers. The oligo(dT)₁₈ anneals selectively on the poly(A) tail of mRNA. Random hexamer primers do not require the presence of poly(A). Therefore, they can be used for transcription of the 5-end regions of mRNA or cDNA synthesis using RNA without poly(A) tail e.g. micro RNAs. Gene-specific primers may also be used with the kits.

The first strand of cDNA can be directly used as a template in PCR (see RT-PCR protocol), real-time PCR or in second strand cDNA synthesis (see protocol). 

Quality Control

The kit is functionally tested in RT-PCR using 100 fg control GAPDH RNA and GAPDH control primers generated a 496 bp product visible on agarose gel after ethidium bromide staining.

Components

·         RevertAid™ Reverse Transcriptase

·         RiboLock™ RNase Inhibitor

·         5X Reaction Buffer

·         dNTP Mix,10 mM each

·         Oligo(dT)₁₈ Primer

·         Random Hexamer Primer

·         Control GAPDH RNA

·         Forward GAPDH Primer, 10 µM (5-CAAGGTCATCCATGACAACTTTG-3)

·         Reverse GAPDH Primer, 10 µM (5-GTCCACCACCCTGTTGCTGTAG-3)

·         Water, nuclease free

·         Detailed Protocol

反转录试剂盒

产品信息

全长的第一链cDNA产量高，最长可合成13 kb的cDNA。

增加的反应温度范围：42-50°C。

配套提供RiboLock™ RNase Inhibitor。

完全一试剂盒提供oligo(dT)18和随机六聚体引物。

应用  

第一链cDNA合成，作为RT-PCR和实时RT-PCR模板(1, 2)。

构建全长cDNA文库。

制备杂交用探针。

aRNA合成。 

说明

RevertAid™ First Strand cDNA Synthesis Kit是一个完整的以RNA为模板高效合成第一链cDNA的反应体系。该试剂盒可合成长达13 kb的cDNA。

该试剂盒使用的RevertAidReverse Transcriptase具有的RNase H 活性比AMV reverse transcriptase低。

试剂盒配套提供的重组RiboLock™ RNase Inhibitor有效保护RNA模板免于降解。该产品与逆转录反应完全兼容，因为其在50°C时仍然保持活性。

试剂盒配套提供oligo(dT)18和随机六聚体引物。oligo(dT)18与mRNA 的poly(A)尾选择性结合。随机六聚体引物转录无需poly(A) 尾，因此可转录mRNA的5’-端区域，也可以无poly(A) 尾的RNA (micro RNAs)为模板合成cDNA。试剂盒也可使用基因特异性引物。

第一链cDNA可直接作为PCR (见RT-PCR操作说明书)、实时PCR或第二链cDNA 合成(见操作说明书)的模板。 

质量控制

功能测试：以多聚腺苷酸RNA转录子为模板合成第一链cDNA(分别测试特异性引物、oligo(dT)18和随机六聚体引物)。合成的cDNA作为后续PCR的模板。 

组分

RevertAid™ Reverse Transcriptase

RiboLock™ RNase Inhibitor

5X Reaction Buffer

dNTP Mix,10 mM each

Oligo(dT)18 Primer

Random Hexamer Primer

Control GAPDH RNA

Forward GAPDH Primer, 10 µM (5-CAAGGTCATCCATGACAACTTTG-3)

Reverse GAPDH Primer, 10 µM (5-GTCCACCACCCTGTTGCTGTAG-3)

Water, nuclease free

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