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

Efficient Preparation of cDNA: PrimeScript Reverse Transcriptase

PrimeScript Reverse Transcriptase has exceptionally strong strand displacement activity and enables efficient preparation of cDNA up to 12 kb in length. It is robust, versatile and well-suited for applications requiring full-length cDNA such as preparation of cDNA libraries and other techniques involving first strand cDNA synthesis (RT-PCR, preparation of cDNA probes, real-time quantitative RT-PCR). PrimeScript RTase can be used for performing a reverse transcription reaction with any RNA template including GC-rich templates and RNAs with high levels of secondary structure. This enzyme is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and is verified to be RNase H Minus. Because of the excellent extension capability of PrimeScript Reverse Transcriptase, preparation of cDNA 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.

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.

  At-A-Glance   Documents   Images & Data   Resources

Features

  • Strong strand displacement and extension capability: Capable of synthesizing long and full-length cDNA molecules (up to 12 kb)
  • Preparation of cDNA at 42°C results in lower background and higher cDNA yields because the lower temperature 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 GC-rich templates and templates with high levels of secondary structure
  • Highly sensitive: use less of your precious RNA samples
  • Demonstrated success with reverse transcription reaction times of 30 min. (60 min. for longer transcripts)

Applications

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

Standard Protocol

  1. Prepare the following mixture in a microtube.
    Oligo dT primer*50 pmol
    dNTP Mixture (10 mM each)1 µl
    Template RNA≤5 µg (total RNA) or ≤1 µg (mRNA)
    RNase-free dH2Oto 10 µl

    * For Random Primers (6-mers), use 50 pmol. For gene-specific primers, use 2 pmol.

  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 Units
    RNase-free dH2Oto 20 µl
  4. Mix gently.
  5. Perform the reaction under the following conditions.
    30°C10 min.*
    42°C**30–60 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 for 30 min. 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.

Components

Cat. # 2680A:

PrimeScript Reverse Transcriptase10,000 U
5X PrimeScript Buffer500 µl

Buffer Composition

5X PrimeScript Buffer (for cDNA synthesis)
250 mMTris-HCl, pH 8.3
375 mMKCl
15 mMMgCl2

Purity

Nuclease activity is not detected in any of the following cases, as judged from the agarose gel electrophoresis pattern:

  1. After incubation of 1 µg of lambda DNA-Hind III fragments with 200 units of this enzyme for 1 hour at 37°C.
  2. After incubation of 1 µg of supercoiled pBR322 DNA with 200 units of this enzyme for 1 hour at 37°C.
  3. After incubation of 1 µg of 16S and 23S rRNA with 200 units of this enzyme for 1 hour at 37°C.

Storage

–20°C

Source

Purified from an E. coli strain expressing the recombinant enzyme.

Unit Definition

One unit is the amount of the enzyme that incorporates 1 nmol of [3H]dTTP in 10 minutes at 37°C, with poly(rA), oligo(dT) 12-18 as the primer-template.

Reaction mixture for unit definition:

  • 50 mM: Tris-HCl, pH 8.3
  • 75 mM: KCl
  • 8mM: MgCl2
  • 10 mM: DTT
  • 20 ug/mL: (ra)n (dT)12-18
  • 0.5 mM: [3H]dTTP
  • 0.1%: NP-40

Storage Buffer Composition

  • 200 mM Tris-HCl, pH 7.8
  • 100 mM NaCl
  • 1 mM EDTA
  • 1 mM DTT
  • 50% Glycerol (v/v)

1st-Strand cDNA Extension Test

According to the standard protocol, 1st strand cDNA was synthesized by incubating 100 ng of total RNA prepared from rat brain and 50 pmol of oligo dT primer with 200 units of PrimeScript Reverse Transcriptase at 42°C for 60 minutes. Then, PCR is performed with this RT reactant as a template. Proper amplification of the 12 kbp target is confirmed by agarose gel electrophoresis.

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 RT-PCR

PrimeScript One-Step RT-PCR Kit, Ver. 2 (Cat. # RR055A and RR055B)




 
 
Products
Cat. # Product Contents Size Price Units Select
2680A PrimeScript™ Reverse Transcriptase 10,000 Units $214.00
2680B PrimeScript™ Reverse Transcriptase 40,000 Units $571.00
 

 

 
 

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