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PrimeScript 1st strand cDNA Synthesis Kit

The PrimeScript 1st strand cDNA Synthesis Kit contains all of the reagents necessary for synthesis of full-length first-strand cDNA from total or poly A⁺ RNA, including both oligo(dT) and random 6-mer primers. This kit is powered by PrimeScript Reverse Transcriptase (PrimeScript RT), which has exceptionally strong strand-displacement activity and efficiently synthesizes cDNA. PrimeScript RT 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 quantitative PCR (qPCR). The PrimeScript 1st 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 RT is now available to scientists worldwide. Over 3,700 peer-reviewed articles have been published in which PrimeScript RT was referenced in a variety of scientific applications, including gene discovery, gene expression and transcriptome analysis, miRNA expression and regulation studies, molecular evolution, virology, and microbiology. PrimeScript RT 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 RT, 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.

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Cat. #	Product	Size	Price
6110A	PrimeScript™ 1st strand cDNA Synthesis Kit	50 Rxns	USD $339.00
The PrimeScript 1st strand cDNA Synthesis Kit contains all the reagents necessary to synthesize first strand cDNA from total or polyA⁺ RNA using PrimeScript RTase, a MMLV (Moloney Murine Leukemia Virus)-derived reverse transcriptase. This enzyme is capable of synthesizing cDNA up to 12 kb in length. PrimeScript RTase can synthesize cDNA efficiently at 42℃, even from RNA templates that contain GC-rich regions or complex secondary structures, making it unnecessary to perform reactions at high temperatures that may degrade RNA. The first strand cDNA synthesized with this kit can be used for variety of applications, including second strand synthesis, hybridization, PCR amplification, and real-time PCR. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components Image Data Resources Back Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase 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 a 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. Back 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 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 first-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 green intercalating dye and fluorescence image analysis. Reactions performed using PrimeScript Reverse Transcriptase showed the highest yield of full-length cDNA product and lowest background. Back 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 Reverse Transcriptase or other commercially available RTases. The template for all reactions was Human Placenta Total RNA (Takara Bio). 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. Back 6110A: PrimeScript 1st strand cDNA Synthesis Kit
6110B	PrimeScript™ 1st strand cDNA Synthesis Kit	200 Rxns	USD $1139.00
The PrimeScript 1st strand cDNA Synthesis Kit contains all the reagents necessary to synthesize first strand cDNA from total or polyA⁺ RNA using PrimeScript RTase, a MMLV (Moloney Murine Leukemia Virus)-derived reverse transcriptase. This enzyme is capable of synthesizing cDNA up to 12 kb in length. PrimeScript RTase can synthesize cDNA efficiently at 42℃, even from RNA templates that contain GC-rich regions or complex secondary structures, making it unnecessary to perform reactions at high temperatures that may degrade RNA. The first strand cDNA synthesized with this kit can be used for variety of applications, including second strand synthesis, hybridization, PCR amplification, and real-time PCR. Cat. # 6110B contains 4 of Cat. # 6110A. Please refer to Cat. # 6110A for complete product documentation and resources. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Image Data Back Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase 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 a 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. Back 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 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 first-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 green intercalating dye and fluorescence image analysis. Reactions performed using PrimeScript Reverse Transcriptase showed the highest yield of full-length cDNA product and lowest background. Back 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 Reverse Transcriptase or other commercially available RTases. The template for all reactions was Human Placenta Total RNA (Takara Bio). 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. Back 6110B: PrimeScript 1st strand cDNA Synthesis Kit

Overview

This cDNA synthesis kit includes oligo dT and random 6-mer primers
Strong strand displacement and extension capability: PrimeScript RT is capable of synthesizing long and full-length cDNA molecules (up to 12 kb)
High specificity: synthesize cDNA at 42°C—a temperature that does not promote RNA degradation—resulting in low background and higher yields
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)

More Information

Applications

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

Components

PrimeScript RT (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 Takara's RNA PCR Kit (AMV) Ver. 3.0. It does not contain the M13 Primer M4 complimentary region.

Standard protocol

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 µ

* 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.
Heat at 65°C for 5 min, then immediately cool on ice.
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 RT 100 to 200 Units

RNase-free dH₂O to 20 µl
Mix gently.
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.
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.

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 1st strand cDNA Synthesis Kit (Cat. # 6110A and 6110B)
Perform 2-step RT-PCR with a kit that includes RT, 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)

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2023 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.

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 µ

Template RNA/Primer Mixture	10 µl
5X PrimeScript Buffer	4 µl
RNase Inhibitor	20 Units
PrimeScript RT	100 to 200 Units
RNase-free dH₂O	to 20 µl

30°C	10 min*
42 to 50°C**	30 to 60 min

PrimeScript 1st strand cDNA Synthesis Kit

Notice to purchaser

Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase

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

Highly accurate reverse transcription with PrimeScript Reverse Transcriptase

6110A: PrimeScript 1st strand cDNA Synthesis Kit