PrimeScript™ 1st strand cDNA Synthesis Kit
 
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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 Primer or Random 6-mers, both included in the kit.) This cDNA synthesis kit is powered by PrimeScript Reverse Transcriptase, which hsa 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 synthesis of cDNA libraries and other techniques involving first strand cDNA synthesis (RT-PCR, preparation of cDNA probes, real-time quantitative RT-PCR). This cDNA synthesis kit can be used for reverse transcription of any RNA template 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 used for a variety of applications such as gene expression, gene discovery, transcriptome analyses, 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.


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


Highly accurate reverse transcription with PrimeScript Reverse Transcriptase Highly accurate reverse transcription with PrimeScript Reverse Transcriptase. First strand cDNA synthesis reactions were performed using PrimeScript ReverseTranscriptase or other commercially available RTases. The template for all reactions was Human Placenta Total RNA (Clontech). All reactions used an oligo-dT primer and were performed according to the recommendations of each manufacturer. After synthesizing cDNA, PCR amplification was performed with highly accurate PrimeSTAR HS DNA Polymerase (target gene: TF, amplification product: 500 bp). A control reaction was included in which PCR was performed (no reverse transcription step). Amplified fragments were cloned and multiple clones from each reaction were sequenced. Error rate was defined as the number of errors per total bases sequenced for each reaction type (~200,000 bases). With only 7 errors out of 201,297 bases (0.0035% error rate), PrimeScript Reverse Transcriptase showed the highest accuracy of all RTases studied.

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cDNAsynthesis with low background, excellent extension, and high yield at 42 degrees C byPrimeScript Reverse Transcriptase  cDNA synthesis with low background, excellent extension, and high yield at 42°C by PrimeScript Reverse Transcriptase in comparison to six other commercially available RTases. RNA ladder (1 kb, 2 kb, 4.4 kb, 6.4 kb, 8.4 kb, 10 kb, and 12 kb) was used as template for 1st strand cDNA synthesis reactions. Reactions were performed at 42°C according to the recommendations of each manufacturer. Equivalent reaction volumes were analyzed using alkaline denaturing gel electrophoresis and products were detected with SYBR Green II and fluorescence image analysis. Reactions performed using PrimeScript Reverse Transcriptase showed the highest yield of full-length cDNA product and lowest background.

Stranddisplacement and elongation activity of PrimeScript Reverse Transcriptase Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase. Top panel: diagram of cDNA synthesis reactions performed using either an oligo dT primer (resulting in 6.4 kb product) or a gene-specific primer (yielding a 4.4 kb product). Primer extension reactions were performed at 50°C. Bottom panel: Primer extension reactions were analyzed on an alkaline denaturing gel. PrimeScript Reverse Transcriptase provided higher yield of both the 6.4 kb and the 4.4 kb product, demonstrating superior strand displacement capability.

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