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

Thermo Scientific™ Maxima H Minus First Strand cDNA Synthesis Kit

Catalog No. FERK1651
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Kit with dsDNase
No. of Reactions:
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20 Reactions
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Catalog No. Includes No. of Reactions
FERK1651 Kit only 20 Reactions
FERK1652 Kit only 100 Reactions
FERK1681 Kit with dsDNase 20 Reactions
FERK1682 Kit with dsDNase 100 Reactions
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Catalog No. FERK1651 Supplier Thermo Scientific™ Supplier No. K1651
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Description

Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit, available with or without dsDNase, is a complete system for highly efficient synthesis of first strand cDNA.

Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit, available with or without dsDNase, is a complete system for highly efficient synthesis of first strand cDNA. The kit uses the Maxima H Minus Reverse Transcriptase (RT), which is an advanced enzyme derived by in vitro evolution of MMLV RT. The enzyme features the highest thermostability among the derivatives of MMLV RT and lacks RNase H activity.

This kit allows synthesis of long cDNAs up to 20 kb at elevated temperatures (up to 65°C), superseding other systems' abilities to produce full-length cDNA. Due to increased synthesis rates the reaction can be completed in 30 minutes.

For reverse transcription the kit uses Maxima H Minus Reverse Transcriptase (RT), which is an advanced enzyme derived by in vitro evolution of MMLV RT.

The Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase provides a dramatically simplified workflow that combines genomic DNA elimination and cDNA synthesis into one-tube procedure. The kit contains a novel double-strand-specific DNase (dsDNase) engineered to remove contaminating genomic DNA from RNA preps in two minutes without damage to quality or quantity of RNA. Highly specific dsDNase activity towards double-stranded DNA ensures that single-stranded DNA (such as cDNA and primers) is not cleaved, and dsDNase treated RNA can be directly added to reverse transcription.

Features of the Maxima H Minus First Strand cDNA Synthesis Kit include:
• Increased reaction temperatures—the first strand of cDNA can be synthesized within the 42–65°C temperature range
• High yields of full-length first strand cDNA—with RNA templates up to 20 kb
• Flexible priming—oligo(dT)18, random hexamer or gene-specific primers
• Integrated genomic DNA removal step with dsDNase kit

Applications
• First Strand cDNA synthesis for RT-PCR
• Construction of cDNA libraries
• Generation of probes for hybridization
• Antisense RNA synthesis

Contents
• Maxima H Minus Enzyme Mix
• Oligo(dT)18 and Random hexamer primers
• 5X RT Buffer
• dNTP Mix
• Nuclease-free water

First Strand Synthesis Kit with dsDNase also contains
• dsDNase and 10X dsDNase Buffer

Additional information about reaction components
• Maxima H Minus Enzyme Mix contains Maxima H Minus Reverse Transcriptase and RiboLock RNase Inhibitor. RiboLock RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B, and C at temperatures up to 55°C.
• Oligo(dT)18 and random hexamer primers are supplied with the kit. Random hexamer primers bind non-specifically and are used to synthesize cDNA from all RNAs in a total RNA population. The oligo(dT)18 primer selectively anneals to the 3'-end of poly(A) RNA, synthesizing cDNA only from poly(A) tailed mRNA. Gene-specific primers may also be used with the kit to prime synthesis from a specified sequence.
• 10 mM dNTP Mix is a premixed aqueous solution of dATP, dTTP, dCTP, and dGTP.
• Nuclease-free water is provided for reaction set-up and dilution of sample DNA. The absence of endo-, exodeoxyribonucleases, ribonucleases, and phosphatases has been confirmed by appropriate quality tests.

Specifications

Content And Storage

• Maxima H Minus Enzyme Mix
• Oligo(dT)18 and Random hexamer primers
• 5X RT Buffer
• dNTP Mix
• Nuclease-free water

Store at –20°C.
Format Kit
GC-Rich PCR Performance High
Reaction Speed 30 min.
Technique Reverse Transcription
Optimal Reaction Temperature 50°C to 55°C
Quantity Each
Reverse Transcriptase Maxima 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 20 kb
Starting Material RNA
Includes Kit only
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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.

Is it preferable to use elevated temperature in the RT reaction when using Maxima reverse transcriptases?

cDNA synthesis at higher temperatures ensures successful transcription of RNA with high levels of secondary structure, reducing issues of primer access to template. Therefore, we do recommend to use RT enzymes with high thermostability, e.g. Maxima and Maxima H Minus Reverse Transcriptases, which provide higher yields of full-length cDNA, better sensitivity, and successful transcription of GC-rich templates.
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.
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.