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PrimeScript First Strand cDNA Synthesis Kit

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The PrimeScript First Strand cDNA Synthesis Kit contains all of the reagents necessary for synthesis of first-strand cDNA from total or poly(A)+ RNA using PrimeScript Reverse Transcriptase. Choose this cDNA synthesis kit when you want to synthesize full-length cDNA with provided primers (select from oligo dT or random 6-mer primers, both included in the kit.) This cDNA synthesis kit is powered byPrimeScript Reverse Transcriptase, which has exceptionally strong strand displacement activity and efficiently synthesizes cDNA. PrimeScript RTase is robust, versatile and well-suited for ;applicationsrequiring full-length cDNA such as cDNA library construction. This PrimeScript-based cDNA synthesis kit also works well with other cDNA-dependent techniques including RT-PCR, cDNA probe preparation and real-time quantitative RT-PCR (qRT-PCR or RT-qPCR). The PrimeScript First Strand cDNA Synthesis Kit is ideal for reverse transcription of many different RNA templates, including GC-rich templates and RNAs with high levels of secondary structure. 


Technical Data:

Applications

  • cDNA synthesis kit for RT-PCR
  • First strand cDNA synthesis
  • cDNA probe preparation
  • Synthesis of cDNA libraries with a high proportion of full-length cDNAs

Features

  • This cDNA synthesis kit includes oligo-dT and random 6-mer primers
  • Strong strand displacement and extension capability: PrimeScript RTase is capable of synthesizing long and full-length cDNA molecules (up to 12 kb)
  • High specificity: synthesize cDNA at 42°C – low background, high yield, and high quality RNA at a temperature that does not promote RNA degradation
  • Outstanding accuracy: Lowest error rate among five commercially available reverse transcriptases tested
  • Easy to use: Low rates of non-specific annealing, even on incompletely denatured RNA
  • Works on challenging templates: Excellent results even with templates that are GC-rich or have high levels of secondary structure
  • Highly sensitive: use less of your precious RNA samples
  • Demonstrated success with RT reaction times of 30 min. and 60 min (for longer transcripts).

Components

PrimeScript RTase (200 U/µL) 50 µL
5X PrimeScript Buffer 200 µL
RNase Inhibitor (40 U/µL) 25 µL
dNTP Mixture (10 mM each) 50 µL
Oligo dT Primer (50 µM) 50 µL
Random 6 mers (50 µM) 100 µL
RNase free H2O 1 mL

Primer Sequences

  • Randon 6 mers: pd(N)6
  • Oligo dT Primer: Takara Bio proprietary dT sequence region*

*This sequence is different from the Oligo dT Adaptor Primer supplied with the TaKara RNA PCR Kit (AMV) Ver. 3.0. It does not contain the M13 Primer M4 complimentary region.

Standard Protocol

  1. Prepare the following mixture in a microtube.
    Oligo dT Primer (50 µM)* 1 µl
    dNTP Mixture (10 mM each) 1 µl
    Template RNA ≤5 µg (total RNA) or ≤1 µg (mRNA)
    RNase-free dH2O to 10 µl

    * For Random 6-mers, use 0.4 µl (20 pmol) for synthesis of cDNA products over 2 kb long, or 2 µl (100 pmol) for reverse transcription prior to real time PCR. For gene-specific primers, use primers at a final concentration of 0.1 µM.

  2. Heat at 65°C for 5 min., then immediately cool on ice.
  3. Prepare the reaction mixture by combining the following components to create a 20-µl reaction.
    Template RNA/Primer Mixture 10 µl
    5X PrimeScript Buffer 4 µl
    RNase Inhibitor 20 Units
    PrimeScript Reverse Transcriptase 100 to 200 Units
    RNase-free dH2O to 20 µl
  4. Mix gently.
  5. Perform the reaction under the following conditions.
    30°C 10 min.*
    42 to 50°C** 30 to 60 min.

    * This step is required for random primers.

    ** PrimeScript RTase has robust extension capability even with template having complex secondary structure. Therefore, incubation at 42°C is generally recommended. However, for RT-PCR reactions where the reverse primer for PCR is also used as a reverse transcription primer, we recommend performing the reverse transcription reaction at 50°C to reduce the possibility of non-specific amplification products.

  6. Heat at 70°C for 15 min. and cool on ice.The resulting product can be used for a 2nd strand synthesis reaction or as a template for PCR.

Storage

-20°C

Choosing a PrimeScript Kit for Endpoint RT-PCR

If you want to…. Use this….
Prepare full-length cDNA using your own primers PrimeScript Reverse Transcriptase (Cat. # 2680A and 2680B)
Prepare full-length cDNA using a kit that includes primers PrimeScript First Strand cDNA Synthesis Kit (Cat. # 6110A and 6110B)
Perform 2-step RT-PCR with a kit that includes RTase, primers, and PCR enzyme PrimeScript RT-PCR Kit (Cat. # RR014A and RR014B)
Perform cDNA cloning studies with extremely high fidelity PrimeScript High Fidelity RT-PCR Kit (Cat. # R022A and R022B)
Perform 1-step PCR PrimeScript  One-Step RT-PCR Kit, Ver. 2 (Cat. # RR055A and RR055B)


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