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Products >  PCR_Products >  RT-PCR-Two_Step >  MMLV_Reverse_Transcriptase >  PrimeScript_First_Strand_cDNA_Synthesis_Kit

cDNA synthesis kit: PrimeScript First Strand cDNA Synthesis Kit

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 by PrimeScript Reverse Transcriptase, which has exceptionally strong strand displacement activity and efficiently synthesizes cDNA. PrimeScript RTase is robust, versatile and well-suited for ;applications requiring 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. 

First introduced in Japan, PrimeScript is now available to scientists worldwide. Over 3,700 peer-reviewed articles have been published in which PrimeScript Reverse Transcriptase was referenced in a variety of scientific applications such as gene expression, gene discovery, transcriptome analysis, miRNA expression and regulation, molecular evolution, virology and microbiology. PrimeScript RTase is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and is verified to have low levels of RNase H. Because of the excellent extension capability of PrimeScript RTase, cDNA synthesis reactions can be performed at a lower temperature (42°C), decreasing the risk of RNA degradation that can occur during conventional reactions performed at higher temperatures.

At-A-Glance Documents Images & Data Resources

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

Components

PrimeScript RTase (200 U/µL)50 µL
5X PrimeScript Buffer200 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 H2O1 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 dH2Oto 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 Mixture10 µl
    5X PrimeScript Buffer4 µl
    RNase Inhibitor20 Units
    PrimeScript Reverse Transcriptase100 to 200 Units
    RNase-free dH2Oto 20 µl
  4. Mix gently.
  5. Perform the reaction under the following conditions.
    30°C10 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)




 
 
Products
Cat. # Product Package Size Price # of Units Select
6110A PrimeScript™ 1st strand cDNA Synthesis Kit 50 Rxns $235.00
6110B PrimeScript™ 1st strand cDNA Synthesis Kit 200 Rxns $798.00
 

 


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