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  7. Thermo Scientific™ RevertAid H Minus First Strand cDNA Synthesis Kit

Thermo Scientific™ RevertAid H Minus First Strand cDNA Synthesis Kit

Catalog No. FERK1631
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FERK1632 100 Reactions
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Catalog No. FERK1631 Supplier Thermo Scientific™ Supplier No. K1631
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Description

Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates.

Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit is a complete system for efficient synthesis of first strand cDNA from RNA templates. The kit includes RevertAid H Minus Reverse Transcriptase, which has a point mutation that completely eliminates RNase H activity. Therefore, it does not degrade RNA in RNA-DNA hybrids during synthesis of the first strand cDNA.

Features of the RevertAid H Minus First Strand cDNA Synthesis Kit include:
• High yields of full-length first-strand cDNA up to 13 kb
• Increased reaction temperatures of 42–55°C
• Supplied with the recombinant RiboLock RNase Inhibitor
• Complete kit includes oligo(dT)18 and random hexamer primers

Applications
• First strand cDNA synthesis for RT-PCR and RT-qPCR
• Construction of full length cDNA libraries
• Antisense RNA synthesis

Additional Features
The recombinant RiboLock RNase Inhibitor, supplied with the kit, effectively protects RNA template from degradation. It is fully compatible with the 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 primer anneals selectively to 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 for cDNA synthesis using RNA without the poly(A) tail, e.g., microRNAs. Gene-specific primers may also be used with the kit. The first strand cDNA can be directly used as a template in PCR, real-time PCR, or in second strand cDNA synthesis.

Specifications

Content And Storage

• RevertAid H Minus Reverse Transcriptase
• RiboLock RNase Inhibitor
• 5X Reaction Buffer
• dNTP Mix
• Oligo(dT)18 Primer
• Random Hexamer Primer
• Control RNA
• Control Primers
• Nuclease-free water

Store at –20°C.
Format Kit
Reaction Speed 60 min.
Technique Reverse Transcription
Optimal Reaction Temperature 42°C to 45°C
Quantity 20 reactions
Reverse Transcriptase RevertAid H Minus
Ribonuclease H Activity Reduced
Shipping Condition Dry Ice
For Use With (Application) Real Time PCR (qPCR), RT-PCR
Final Product Type First-Strand cDNA
No. of Reactions 20 Reactions
Reaction Format Separate components
Reagent Type Reverse Transcription
Size (Final Product) Up to 13 kb
Starting Material RNA
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Frequently Asked Questions (FAQs)

When should I choose regular RevertAid RT or Maxima RT vs. RevertAid H-minus RT or Maxima H-minus RT?

It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. We also recommended using RNase H-minus RTs for template-independent addition of C nucleotides.

Do Thermo Scientific reverse transcriptases (RevertAid RT, RevertAid H-minus RT, Maxima RT, and Maxima H-minus RT) possess terminal deoxynucleotidyl (TdT) activity?

All Thermo Scientific reverse transcriptases possess intrinsic TdT activity although at varying degrees depending upon the reaction conditions. For addition of template-independent C nucleotides (as for SMART and RACE experiments), this specific TdT activity can be induced by Mn2+. We would recommend Maxima H- or RevertAid H- minus RTs for this purpose.
What steps should I take while performing first strand cDNA synthesis using low purity template (e.g., inhibitors in RNA sample)?

Trace amounts of reagents used in RNA purification protocols may remain in solution and inhibit first-strand synthesis, e.g., SDS, EDTA, guanidine salts, phosphate, pyrophosphate, polyamines, spermidine. To remove trace contaminants, we recommend re-precipitating the RNA with ethanol and washing the pellet with 75% ethanol, or re-purifying the RNA.
For reverse transcription, how important is the quality of RNA template?

RNA purity and integrity are essential for synthesis and quantification of cDNA. Always assess the integrity of RNA prior to cDNA synthesis. Use freshly prepared RNA. Multiple freeze/thaw cycles of the RNA sample and synthesized cDNA is not recommended. Avoid RNase contamination and discard low quality RNA.
When should I choose regular RevertAid RT or Maxima RT vs. RevertAid H Minus RT or Maxima H Minus RT?

It is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning. RNase H degrades RNA from RNA-DNA duplexes, which can result in truncated cDNA during reverse transcription of long mRNA. We also recommended using RNase H Minus RTs for template-independent addition of C nucleotides. In contrast, reverse transcriptases with intrinsic RNase H activity are often favored in 1-step RT-qPCR applications.
What is the fidelity of RevertAid and Maxima reverse transcriptases?

The fidelity of RevertAid and Maxima reverse transcriptases is the same as that of wild-type M-MuLV RT, which is in the range of 1 error per 15,000-27,000 nucleotides synthesized.
Do Thermo Scientific reverse transcriptases (RevertAid RT, RevertAid H Minus RT, Maxima RT, and Maxima H Minus RT) possess terminal deoxynucleotidyl (TdT) activity?

All Thermo Scientific reverse transcriptases (RevertAid RT, RevertAid H Minus RT, Maxima RT, and Maxima H Minus RT) possess intrinsic TdT activity, although at varying degrees depending upon the reaction conditions. We recommend specialized SuperScript IV Template Switching RT Master Mix for high efficiency in applications requiring template switching RT.

For Research Use Only. Not for use in diagnostic procedures.