Thermo Scientific™ Maxima First Strand cDNA Synthesis Kit for RT-qPCR

Produktkode	Includes	No. of Reactions	unitSize
15279064	Kit with dsDNase	50 Reactions	50 reactions
10282650	Kit only	50 Reactions	50 reactions
10334500	Kit only	200 Reactions	200 reactions
15273796	Kit with dsDNase	200 Reactions	200 reactions

Produktkode 15279064 missing translation for 'mfr' Thermo Scientific™ missing translation for 'supplierNo' K1671

Beskrivelse

The Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR, available with or without dsDNase, is a convenient system optimized for cDNA synthesis in two-step quantitative RT-PCR (RT-qPCR) applications.

The Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR, available with or without dsDNase, is a convenient system optimized for cDNA synthesis in two-step quantitative RT-PCR (RT-qPCR) applications. It provides a dramatically simplified workflow that combines genomic DNA elimination and cDNA synthesis into one-tube procedure. These cDNA Synthesis kits use Maxima Reverse Transcriptase (RT), an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features high thermostability, robustness and increased cDNA synthesis rate compared to wild type M-MuLV RT.

The Maxima First Strand cDNA Synthesis Kit for RT-qPCR is capable of reproducible cDNA synthesis from a wide range of total RNA amounts (1 pg to 5 μg) at elevated temperatures (42–65°C). The synthesis reaction can be completed in 15–30 minutes. Components of the Maxima First Strand cDNA Synthesis Kit for RT-qPCR are pre-mixed to save time and to reduce the possibility of pipetting errors.

The Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase also contains a novel double-strand specific DNase engineered to remove contaminating genomic DNA from RNA preps in two minutes without damage to quality or quantity of RNA. Highly specific 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 First Strand cDNA Synthesis Kit for RT-qPCR include:
• High yields of full length cDNA up to 20 kb
• Efficient cDNA synthesis at wide temperature range (42–65°C)
• Increased synthesis rate—complete cDNA synthesis in 15–30 minutes
• High sensitivity and specificity
• Integrated gDNA removal step with dsDNase kit

Maxima First Strand cDNA Synthesis Kit with dsDNase contains:
• Maxima Enzyme Mix
• 5X Reaction Mix
• nuclease-free water

Kit with dsDNase also contains
• dsDNase
• 10X dsDNase Buffer

Applications
• 2-step RT-PCR
• 2-step RT-qPCR

Additional information about reaction components
The Maxima Enzyme Mix contains Maxima Reverse Transcriptase and RiboLock RNase Inhibitor. The recombinant RiboLock RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B, and C at temperatures up to 55°C.

5X Reaction Mix contains the remaining reaction components: reaction buffer, dNTPs, oligo(dT)18 and random hexamer primers. Nuclease-free water is provided for reaction set-up and dilution of sample RNA.

The absence of endo- and exo-deoxyribonucleases, ribonucleases, and phosphatases has been confirmed by appropriate quality tests.

Tekniske data

• Content And Storage:

  • Maxima Enzyme Mix
  • 5X Reaction Mix
  • dsDNase
  • 10X dsDNase Buffer
  • 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
• Ribonuclease H Activity: Yes
• Shipping Condition: Dry Ice
• For Use With (Application): Real Time PCR (qPCR)
• Final Product Type: First-Strand cDNA
• No. of Reactions: 50 Reactions
• Reaction Format: Separate components
• Reagent Type: Reverse Transcription
• Size (Final Product): Up to 20 kb
• Starting Material: RNA
• Includes: Kit with dsDNase

Frequently Asked Questions (FAQs)

How can I inactivate Thermo Scientific dsDNase?

dsDNase can be inactivated by incubating the sample at 55°C for 5 min in the presence of 10 mM DTT.

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.

Does dsDNase cleave ssDNA?

No, dsDNase cleaves only double-stranded DNA. However, if ssDNA forms double-stranded structures, it will act as a target for dsDNase. Because of this, long and complex ssDNA may be partially degraded. Therefore we recommend additional dsDNase inactivation step for RT-PCR amplification of targets 3 kb or longer. The inactivation step should be performed by 5 min incubation at 55 degrees C in the presence of 10 mM DTT.

Can dsDNase be added directly to a reverse transcription reaction to remove genomic DNA?

No. RT reaction composition inhibits dsDNase activity, and genomic DNA removal may be incomplete.

When using the Maxima or Maxima H Minus First Strand cDNA Synthesis kits with dsDNase, can the genomic DNA elimination step be omitted?

Yes. dsDNase buffer is not needed for subsequent reverse transcription reaction. If RNA sample is not contaminated with gDNA, dsDNase treatment may be omitted.

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.

How does the dsDNase in Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase (Cat. Nos. K1671 and K1672) compare with TURBO DNase?

The main difference between the two enzymes is that dsDNase is heat-labile (easily inactivated by moderate heat treatment at 55 degrees C) and specific to dsDNA. Therefore, it does not need a separate heat inactivation step. It gets inactivated during the reverse transcription reaction. Turbo DNase is not heat-labile and is able to degrade both ssDNA and dsDNA. It needs to be heat inactivated post-digestion or has to be removed by phenol-chloroform extraction.

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.