cDNA synthesis kits

Complete kit for the synthesis of first-strand cDNA from total RNA or polyA⁺ RNA. The kit contains sufficient reagents for 25 first-strand cDNA synthesis reactions.

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The reliability of cDNA synthesized with the Advantage RT-for-PCR Kit

The reliability of cDNA synthesized with the Advantage RT-for-PCR Kit. The time course of lipopolysaccharide induction of iNOS mRNA in the mouse macrophage cell line RAW 264.7 was studied by isolating RNA from culture samples taken at the time points indicated (in hr). Approx. 0.4 μg was then reverse transcribed using the Advantage RT-for-PCR Kit, and 5% of the cDNA product was used as a template for PCR using the Mouse G3PDH Amplimer Set (Cat. # 639009) as a control (Panel A) and mouse iNOS primers (Panel B). 20% portions of the PCR products were run on a 1.8% EtBr/agarose gel. Lane M: ΦX174/Hae III DNA size marker.

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PCR amplification of cDNA synthesized with the Advantage RT-for-PCR Kit vs. cDNA synthesized with kits from two competitors

PCR amplification of cDNA synthesized with the Advantage RT-for-PCR Kit vs. cDNA synthesized with kits from two competitors. The indicated amounts of Human Lung Total RNA (Cat. # 636507) were used for reverse transcription, and 5% of each cDNA product was amplified by PCR using the Human G3PDH Control Amplimer Set (Cat. # 639005). Lanes 1, 5 & 9: 250 ng. Lanes 2, 6 & 10: 62.5 ng. Lanes 3, 7 & 11: 16 ng. Lanes 4, 8 & 12: 4 ng. Lane M: Φ X174/Hae III DNA size marker. The results show that when 4 ng of RNA was used as the starting material, only the Advantage RT-for-PCR Kit generated a clearly visible product.

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639505: Advantage RT-for-PCR Kit

Complete kit for the synthesis of first-strand cDNA from total RNA or polyA⁺ RNA. The kit contains sufficient reagents for 100 first-strand cDNA synthesis reactions.

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The reliability of cDNA synthesized with the Advantage RT-for-PCR Kit

The reliability of cDNA synthesized with the Advantage RT-for-PCR Kit. The time course of lipopolysaccharide induction of iNOS mRNA in the mouse macrophage cell line RAW 264.7 was studied by isolating RNA from culture samples taken at the time points indicated (in hr). Approx. 0.4 μg was then reverse transcribed using the Advantage RT-for-PCR Kit, and 5% of the cDNA product was used as a template for PCR using the Mouse G3PDH Amplimer Set (Cat. # 639009) as a control (Panel A) and mouse iNOS primers (Panel B). 20% portions of the PCR products were run on a 1.8% EtBr/agarose gel. Lane M: ΦX174/Hae III DNA size marker.

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PCR amplification of cDNA synthesized with the Advantage RT-for-PCR Kit vs. cDNA synthesized with kits from two competitors

PCR amplification of cDNA synthesized with the Advantage RT-for-PCR Kit vs. cDNA synthesized with kits from two competitors. The indicated amounts of Human Lung Total RNA (Cat. # 636507) were used for reverse transcription, and 5% of each cDNA product was amplified by PCR using the Human G3PDH Control Amplimer Set (Cat. # 639005). Lanes 1, 5 & 9: 250 ng. Lanes 2, 6 & 10: 62.5 ng. Lanes 3, 7 & 11: 16 ng. Lanes 4, 8 & 12: 4 ng. Lane M: Φ X174/Hae III DNA size marker. The results show that when 4 ng of RNA was used as the starting material, only the Advantage RT-for-PCR Kit generated a clearly visible product.

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639506: Advantage RT-for-PCR Kit

The BcaBEST RNA PCR Kit is designed to perform both reverse transcription and DNA amplification in a single tube. The kit uses BcaBEST DNA Polymerase, which contains both DNA polymerase and reverse transcriptase for first strand cDNA synthesis. In contrast to standard reverse transcriptases, BcaBEST Polymerase has a temperature optimum of 65°C, which permits cDNA synthesis from difficult and highly structured RNA templates. Bca-Optimized Taq Polymerase is used for second strand synthesis and subsequent PCR. This polymerase utilizes LA PCR technology for improved PCR length and accuracy. Random 9-mers, oligo-dT primers, or a specific downstream primer that acts as an antisense primer in PCR can be used for cDNA synthesis.

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Amplification of human TRADD gene

Amplification of human TRADD gene. Amplification of the human TRADD (TNF receptor associated death domain) gene (494 bp, GC content 68.2%) was performed using total RNA from HeLa cells. Lanes 1 and 2 used the RNA PCR Kit, Ver. 2.1, and the BcaBest; RNA PCR Kit, Version 1.1, respectively. Lanes M contain a 100-bp ladder.

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RR023A: BcaBEST RNA PCR Kit Ver. 1.1

The BcaBEST RNA PCR Kit is designed to perform both reverse transcription and DNA amplification in a single tube. The kit uses BcaBEST DNA Polymerase, which contains both DNA polymerase and reverse transcriptase for first strand cDNA synthesis. In contrast to standard reverse transcriptases, BcaBEST Polymerase has a temperature optimum of 65°C, which permits cDNA synthesis from difficult and highly structured RNA templates. Bca-Optimized Taq Polymerase is used for second strand synthesis and subsequent PCR. This polymerase utilizes LA PCR technology for improved PCR length and accuracy. Random 9-mers, oligo-dT primers, or a specific downstream primer that acts as an antisense primer in PCR can be used for cDNA synthesis. Cat. # RR023B contains 2 of Cat. # RR023A. Please refer to Cat. # RR023A for complete product documentation and resources.

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RR023B: BcaBEST RNA PCR Kit Ver. 1.1

Complete kit for comparing two bacterial genomes (from two different species of bacteria or two different strains of the same species) and obtaining DNA fragments that are present in one genome but not in the other. Enough reagents are provided for one control and six complete subtractions.

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Differences in gene content between unrelated H. pylori strains

Differences in gene content between unrelated H. pylori strains. Two unrelated H. pylori strains were used for PCR-Select subtraction. J166 was used as tester; 26695 was used as driver. After amplification by PCR, unsubtracted amplified tester (Lane 1) and driver (Lane 2) samples were electrophoresed on a 1.5% agarose gel and transferred onto nylon filters. These filters were hybridized with randomly picked clones from the cloned subtracted library (Panels A–D). Out of 20 clones analyzed, 10 hybridized only to the tester (e.g., Panels A & B) or hybridized to the tester with higher efficiency (e.g., Panel C).

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637404: Clontech PCR-Select Bacterial Genome Subtraction Kit

Kit for identifying cDNAs that correspond to differentially expressed sequences in one cDNA population compared with another. Enough reagents are provided for seven cDNA reactions. PCR primers are provided for 50 primary and 100 secondary PCR amplifications—enough for one control and six complete subtractions if the cDNA from each synthesis is used for tester and driver in separate subtractions.

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Southern blot analysis showing enrichment of a gene activated in human Jurkat T-cells by PHA/PMA treatment, and reduction of an abundant housekeeping gene in subtracted cDNA

Southern blot analysis showing enrichment of a gene activated in human Jurkat T-cells by PHA/PMA treatment, and reduction of an abundant housekeeping gene in subtracted cDNA. Tester cDNA was prepared from human Jurkat leukemic T-cells and incubated with 2 µg/ml HA and 2 ng/ml PMA for 72 hr. Driver cDNA was prepared from the same untreated cells. Amplified tester, driver, and subtracted cDNA were electrophoresed on a 1.5% agarose gel (0.3 µg per lane), transferred onto nylon filters, and hybridized with either an IL-2R alpha probe, a known marker of activation (Panel A), or a G3PDH housekeeping gene probe (Panel B).

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637401: Clontech PCR-Select cDNA Subtraction Kit

Hybridization Blocking Solution for use with the Clontech PCR-Select Differential Screening Kit.

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The PCR-Select Differential Screening Kit detects rare, differentially expressed cDNAs

The PCR-Select Differential Screening Kit detects rare, differentially expressed cDNAs. The SMART PCR cDNA Synthesis Kit (Cat. # 634902) was used to preamplify total RNA from a gamma-globin-producing cell line and a beta-globin-producing cell line. PCR-Select cDNA subtraction was performed using the gamma-line cDNA as tester and beta-line cDNA as driver; for the reverse subtraction, tester and driver were switched. The subtracted cDNA was then cloned, and randomly selected clones spotted on nylon membranes for duplicate screening. Membranes were hybridized with the indicated probe.

Complete kit for differential screening of subtracted libraries obtained using the Clontech PCR-Select cDNA Subtraction Kit. The differential screening procedure can be used to identify putative differentially expressed sequences in the subtracted library before performing Northern blot analysis. The kit contains reagents for making cDNA probes for differential screening and hybridization control cDNAs.

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The PCR-Select Differential Screening Kit detects rare, differentially expressed cDNAs

The PCR-Select Differential Screening Kit detects rare, differentially expressed cDNAs. The SMART PCR cDNA Synthesis Kit (Cat. # 634902) was used to preamplify total RNA from a gamma-globin-producing cell line and a beta-globin-producing cell line. PCR-Select cDNA subtraction was performed using the gamma-line cDNA as tester and beta-line cDNA as driver; for the reverse subtraction, tester and driver were switched. The subtracted cDNA was then cloned, and randomly selected clones spotted on nylon membranes for duplicate screening. Membranes were hybridized with the indicated probe.

The In-Fusion SMARTer Directional cDNA Library Construction Kit provides a sensitive and efficient method for producing high-quality, full-length cDNA libraries. The kit utilizes two of our most innovative technologies: SMART (Switching Mechanism At 5' end of RNA Transcript) technology coupled with PCR amplification makes it possible to generate high yields of full-length double-stranded cDNA from nanograms of poly A⁺ or total RNA. In-Fusion cloning technology makes it easy to clone your SMARTer cDNA library into any location within any vector. You may use the pSMART2IFD Linearized Vector included in the kit, or any vector of your choice, for cloning your library. Isolated clones from finished libraries can be transferred directly to any linearized expression vector for functional analysis-without the need for compatible restriction sites.

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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The In-Fusion SMARTer Directional cDNA Library Construction Kit includes components for cDNA synthesis, library construction, and library amplification

The In-Fusion SMARTer Directional cDNA Library Construction Kit includes components for cDNA synthesis, library construction, and library amplification.

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Insert size screening by PCR

Insert size screening by PCR. An In-Fusion SMARTer Directional cDNA library was generated from 10 ng of Control Mouse Liver Total RNA. SMARTer ds cDNA was synthesized, size-fractionated, and then cloned into pSMART2IFD using the In-Fusion Cloning Kit. 15 randomly selected colonies from the unamplified library were size screened by PCR according to the protocol in the User Manual. 3-μl samples of PCR products were electrophoresed on a 1.2% agarose/EtBr gel. Lane M: 1-kb DNA ladder.

This kit contains reagents for making adaptor-ligated ds cDNA from poly A+ RNA and performing 5' and 3' Marathon RACE PCR using gene-specific primers and a mixture of thermostable DNA polymerases supplied by the user. This kit contains sufficient reagents for 5 cDNA synthesis reactions and 100 PCR reactions.

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Marathon cDNA amplification of abundant (actin, 1.9 kb) and moderately rare (TFR, 5.1 kb) transcripts. The 5'- and 3'-RACE reactions for actin and TFR were performed with adaptor-ligated ds cDNA made from 1 μg of human placental poly A⁺ RNA and amplified for 25 PCR cycles. Full-length cDNAs were end-to-end PCR–amplified according to the User Manual. Lane 1: 1.2-kb actin 5'-RACE product. Lane 2: 1.3-kb actin 3'-RACE product. Lane 3: full-length 1.9-kb actin cDNA. Lane 4: 2.6- kb TFR 5'-RACE product. Lane 5: 2.9-kb TFR 3'-RACE product. Lane 6: full-length 5.1-kb TFR cDNA. Lane M: 1-kb DNA size ladder.                             

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634913: Marathon cDNA Amplification Kit

Takara Bio's One Step RNA PCR Kit offers the convenience and time savings of performing reverse transcription and PCR in a single tube, without the need for changing buffers or adding reagents, minimizing the risk of contamination. AMV Reverse Transcriptase XL provides greater thermostability than other reverse transcriptases, and retains its activity over a wider range of reaction temperatures. The kit also contains AMV-Optimized Taq DNA Polymerase for efficient amplification of the cDNA synthesized by AMV Reverse Transcriptase XL.

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RT-PCR Amplification from Total RNA using the One Step RNA PCR Kit

RT-PCR Amplification from Total RNA using the One Step RNA PCR Kit. RNA was isolated from HL60 cells and used as a template for RT-PCR with the One-Step RNA PCR Kit. The primers chosen amplified products of 1 kb (lane 1), 2 kb (lane 2), and 4.4 kb (lane 3). Lane M contains lambda-Hind III DNA Markers.

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RR024A: One-Step RNA PCR Kit (AMV)

Takara's One Step RNA PCR Kit offers the convenience and time savings of performing reverse transcription and PCR in a single tube, without the need for changing buffers or adding reagents, minimizing the risk of contamination. AMV Reverse Transcriptase XL provides greater thermostability than other reverse transcriptases, and retains its activity over a wider range of reaction temperatures. The kit also contains AMV-Optimized Taq DNA Polymerase for efficient amplification of the cDNA synthesized by AMV Reverse Transcriptase XL. Cat. # RR024B contains 2 of Cat. # RR024A. Please refer to Cat. # RR024A for complete product documentation and resources.

pEXP-Lib Vector contains an internal ribosome entry site (IRES), which permits the translation of two open reading frames from one messenger RNA. After selection with puromycin, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. SfiIA and SfiIB indicate two distinct SfiI sites that differ in their interpalindromic sequences. This arrangement permits cloning of libraries such that all inserts are in the proper orientation for protein translation.

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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pEXP-Lib vector map

pEXP-Lib vector map. pEXP-Lib is a mammalian expression vector that permits the screening and analysis of libraries directly in mammalian cell lines.

pRetro-Lib, derived from Moloney murine leukemia virus (MoMuLV), is designed for delivery and expression of retroviral libraries or genes. Upon transfection into a packaging cell line, pRetro-Lib can transiently express, or integrate and stably express, a transcript containing (psi, the extended viral packaging signal) and the gene of interest. The 5' viral LTR in this vector contains a viral promoter that controls expression of genes cloned into the MCS.

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pRetro-Lib Vector map

pRetro-Lib Vector map. A retroviral expression vector for cloning libraries. Suitable for use with the SMART cDNA Library Construction Kit (Cat. # 634901).

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.

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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.

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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.

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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.

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6110A: PrimeScript 1st strand cDNA Synthesis Kit

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.

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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.

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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.

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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.

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6110B: PrimeScript 1st strand cDNA Synthesis Kit

cDNA cloning is used for structural analysis of genes and RNAs, and expression of target proteins. Generating a cDNA library involves synthesizing double-stranded cDNA that is complementary to the target mRNA, inserting the cDNA into a vector of bacterial or viral origin, and introducing the vector into bacterial or eukaryotic cells. The cloned cDNA can be then used for analysis, and in vitro transcription and translation. The PrimeScript Double Strand cDNA Synthesis Kit synthesizes double-stranded cDNA from polyA⁺ RNA of animal or plant origin using the Gubler-Hoffman method.

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Double-stranded cDNA synthesis using either oligo(dT) or random primers

Double-stranded cDNA synthesis using either oligo(dT) or random primers.

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6111A: PrimeScript Double Strand cDNA Synthesis Kit

The PrimeScript High Fidelity RT-PCR Kit is designed to synthesize and amplify cDNA from total RNA or mRNA with high fidelity. This kit includes all reagents necessary for reverse transcription of RNA to cDNA followed by PCR amplification of cDNA, including PrimeScript reverse transcriptase and PrimeSTAR Max DNA Polymerase.

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R022A: PrimeScript High Fidelity RT-PCR Kit

The PrimeScript High Fidelity RT-PCR Kit is designed to synthesize and amplify cDNA from total RNA or mRNA with high fidelity. This kit includes all reagents necessary for reverse transcription of RNA to cDNA followed by PCR amplification of cDNA, including PrimeScript reverse transcriptase and PrimeSTAR Max DNA Polymerase. Cat. # R022B contains 4 of Cat. # R022A. Please refer to Cat. # R022A for complete product documentation and resources.

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R022B: PrimeScript High Fidelity RT-PCR Kit

PrimeScript IV 1st strand cDNA Synthesis Mix contains a 5X premix for synthesizing first-strand cDNA from total RNA or poly A+ RNA. A cDNA synthesis reaction can be started simply by adding template RNA and water to the premix, which already contains PrimeScript IV Reverse Transcriptase, RNase Inhibitor, Oligo dT Primer, dNTP Mixture, and reaction buffer. The premix also contains an accessory protein that eliminates the need for RNA denaturation, which is commonly performed before reverse transcription.

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6215A: PrimeScript IV 1st strand cDNA Synthesis Mix

The PrimeScript One Step RT-PCR Kit Ver.2 is designed for RT-PCR in a single tube. All components necessary for RT-PCR are mixed in one tube, and therefore it is unnecessary to add reagents at the midpoint of the procedure and the risk of contamination is minimized. This kit includes PrimeScript 1 step Enzyme Mix (a highly optimized premix of PrimeScript Reverse Transcriptase, which has excellent extension even for RNA templates containing higher-order structure, the high efficiency hot-start PCR enzyme Takara Ex Taq HS, and RNase Inhibitor together with a stabilizing agent) and 2X 1 step buffer (a premix containing reaction buffer, dNTP mixture, and 1 step Enhancer Solution). These premix components allow easy preparation of reaction mixtures. This kit facilitates efficient production of RT-PCR products through efficient reverse transcription at 50°C by permitting reaction conditions and reduction in non-specific amplification that can arise from mispriming or from primer-dimer during pre-cycling steps. This kit includes all reagents necessary for reverse transcription of RNA to cDNA and cDNA amplification using PCR.

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RR055A: PrimeScript One Step RT-PCR Kit Ver.2

The PrimeScript One Step RT-PCR Kit Ver.2 is designed for RT-PCR in a single tube. All components necessary for RT-PCR are mixed in one tube, and therefore it is unnecessary to add reagents at the midpoint of the procedure and the risk of contamination is minimized. This kit includes PrimeScript 1 step Enzyme Mix (a highly optimized premix of PrimeScript Reverse Transcriptase, which has excellent extension even for RNA templates containing higher-order structure, the high efficiency hot-start PCR enzyme Takara Ex Taq HS, and RNase Inhibitor together with a stabilizing agent) and 2X 1 step buffer (a premix containing reaction buffer, dNTP mixture, and 1 step Enhancer Solution). These premix components allow easy preparation of reaction mixtures. This kit facilitates efficient production of RT-PCR products through efficient reverse transcription at 50°C by permitting reaction conditions and reduction in non-specific amplification that can arise from mispriming or from primer-dimer during pre-cycling steps. This kit includes all reagents necessary for reverse transcription of RNA to cDNA and cDNA amplification using PCR. Cat. # RR055B contains 4 of Cat. # RR055A. Please refer to Cat. # RR055A for complete product documentation and resources.

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RR055B: PrimeScript One Step RT-PCR Kit Ver.2

The PrimeScript One Step RT-PCR Kit Ver.2 (Dye Plus) is used for one-step RT-PCR in a single tube. There is no need to add reagents during the reaction, minimizing the risk of contamination. Furthermore, electrophoresis loading dyes (blue and yellow) are included in the premix to allow direct analysis by electrophoresis. The bright green color of the reaction mixture allows visual confirmation of gel loading. The kit includes PrimeScript RTase, a reverse transcriptase that provides superior elongation, and Takara Ex Taq HS, a PCR enzyme formulated for hot start PCR that provides high yields. The reaction components are optimized to provide the same level of performance as conventional RT-PCR systems that lack dyes and loading buffer. This kit includes all of the reagents necessary for the synthesis of cDNA from RNA by reverse transcription and amplification of cDNA by PCR.

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RR057A: PrimeScript One Step RT-PCR Kit, Ver.2 (Dye Plus)

The PrimeScript One Step RT-PCR Kit Ver.2 (Dye Plus) is used for one-step RT-PCR in a single tube. There is no need to add reagents during the reaction, minimizing the risk of contamination. Furthermore, electrophoresis loading dyes (blue and yellow) are included in the premix to allow direct analysis by electrophoresis. The bright green color of the reaction mixture allows visual confirmation of gel loading. The kit includes PrimeScript RTase, a reverse transcriptase that provides superior elongation, and Takara Ex Taq HS, a PCR enzyme formulated for hot start PCR that provides high yields. The reaction components are optimized to provide the same level of performance as conventional RT-PCR systems that lack dyes and loading buffer. This kit includes all of the reagents necessary for the synthesis of cDNA from RNA by reverse transcription and amplification of cDNA by PCR. Cat. # RR057B contains 4 of Cat. # RR057A. Please refer to Cat. # RR057A for complete product documentation and resources.

This kit for two-step RT-PCR is designed to provide excellent extendibility and efficient amplification. It uses PrimeScript reverse transcriptase and Takara Ex Taq HS polymerase. This kit generates RT-PCR products efficiently at standard reverse transcription temperature (42℃), even when RNA templates contain higher-order structures. It is not necessary to perform reactions at a high temperature when there is a risk of RNA decomposition. This kit includes all reagents necessary for the reverse transcription of RNA to cDNA, and for cDNA amplification using PCR.

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RT-PCR using various amounts of RNA template

RT-PCR using various amounts of RNA template. To illustrate the high sensitivity of the PrimeScript™ RT-PCR Kit in detecting mRNAs in very low quantities of total RNA template, GAPDH mRNA was used as a target in various amounts of HL60 cell total RNA (0.1 pg to 1 ng). The RNA was reverse transcribed using the oligo dT primer. The kit protocol was followed for RT and PCR.

Protocol:

Target mRNA	Total RNA
GAPDH (428 nt)	HL60 cell
RT reaction: 0.1 pg –> 1 ng total RNA/20 µl reaction PCR: 2 µl RT products/50 µl reaction

PCR conditions:

94°C –> 1 min.

(40 cycles)

94°C –> 30 sec.

55°C –> 30 sec.

72°C –> 1 min.

Results:

Detection of the GAPDH target mRNA was possible using as little as 1 pg of total RNA.

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Amplifying various lengths of target gene mRNAs by RT-PCR

Amplifying various lengths of target gene mRNAs by RT-PCR. Using human heart total RNA or HL60 cell total RNA as a template, a variety of sequence lengths from two target gene mRNAs (dystrophin and transferrin receptor) were reverse transcribed and amplified by PCR using the reagents in the PrimeScript™ RT-PCR Kit.

 

Protocol:

Target mRNA	Total RNA
Dystrophin	Human Heart
Transferrin receptor (TFR)	HL60 cell

 

Reverse transcription: 1 µg total RNA/20-µl reaction

PCR: 2 µl RT products/50-µl reaction*
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*Corresponds to 100 ng total RNA/50-µl PCR reaction

 

PCR conditions:

0.5~6kb	8~12 kb
94°C –>1 min	94°C –> 1 min
(30 cycles)	(30 cycles)
94°C –> 30 sec.	98°C –> 10 sec
55°C –> 30 sec.	68°C –> 8 or 15 min
72°C –> 1 min./kb

Results

Excellent yields were obtained from all reactions (see figure), confirming efficient extension and amplification of sequences between 0.5 kb and 12 kb in length.

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Principle of PrimeScript RT-PCR Kit

Principle of PrimeScript RT-PCR Kit.

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RR014A: PrimeScript RT-PCR Kit

This kit for two-step RT-PCR is designed to provide excellent extendibility and efficient amplification. It uses PrimeScript reverse transcriptase and Takara Ex Taq HS polymerase. This kit generates RT-PCR products efficiently at standard reverse transcription temperature (42℃), even when RNA templates contain higher-order structures. It is not necessary to perform reactions at a high temperature when there is a risk of RNA decomposition. This kit includes all reagents necessary for the reverse transcription of RNA to cDNA, and for cDNA amplification using PCR. Cat. # RR014B contains 4 of Cat. # RR014A. Please refer to Cat. # RR014A for complete product documentation and resources.

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RR014B: PrimeScript RT-PCR Kit

The RNA LA PCR Kit is designed to generate longer and more accurate RT-PCR products. The kit uses AMV RT XL as the reverse transcriptase, which allows for efficient synthesis of first-strand cDNA up to 12 kb. AMV RT XL also provides better thermostability than other reverse transcriptases, and retains its activity over a wider range of reaction temperatures. In addition, the kit uses LA Taq, which can synthesize products up to 40 kb in size with 6.5X better fidelity than Taq Polymerase. Both the reverse transcription and amplification reactions can be performed in a single tube. The components for 3'-RACE are also supplied.

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Amplification of long mRNA (approx. 12 kb) using the RNA LA PCR Kit

Amplification of long mRNA (approx. 12 kb) using the RNA LA PCR Kit. The RNA LA PCR Kit was used to amplify several long fragments from the dystrophin gene (6, 8, and 12 kb) from human heart mRNA. This data shows excellent performance, even on very long products. Template: Human heart mRNA. Target cDNA: Dystrophin (6, 8, 12 kb).

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Schematic diagram of RNA PCR using the RNA LA PCR Kit, Version 1.1

Schematic diagram of RNA PCR using the RNA LA PCR Kit, Version 1.1.

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RR012A: TaKaRa RNA LA PCR Kit (AMV) Ver.1.1

The RNA PCR Kit (AMV) Version 3.0 is designed for performing single tube reverse transcription of RNA to cDNA using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase, with subsequent cDNA amplification using Takara Ex Taq HS. Taking advantage of AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42–60°C) than MMLV RTase, this kit allows transcription at higher temperatures, which eliminates potential RNA secondary structure that can give rise to polymerase pauses. The supplied Oligo dT-Adaptor Primer is designed for highly efficient cDNA synthesis from the poly(A)⁺ RNA 3'-terminus, which allows amplification of unknown 3'-termini using 3'-RACE. The components for the 3'-RACE method are supplied in the kit.

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Principle of RNA PCR Kit, Version 3.0

Principle of RNA PCR Kit, Version 3.0.

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RR019A: RNA PCR Kit (AMV) Version 3.0

The RNA PCR Kit (AMV) Version 3.0 is designed for performing single tube reverse transcription of RNA to cDNA using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase, with subsequent cDNA amplification using Takara Ex Taq HS. Taking advantage of AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42-60°C) than MMLV RTase, this kit allows transcription at higher temperatures, which eliminates potential RNA secondary structure that can give rise to polymerase pauses. The supplied Oligo dT-Adaptor Primer is designed for highly efficient cDNA synthesis from the poly(A)⁺ RNA 3'-terminus, which allows amplification of unknown 3'-termini using 3'-RACE. The components for the 3'-RACE method are supplied in the kit. Cat. # RR019B contains 2 of Cat. # RR019A. Please refer to Cat. # RR019A for complete product documentation and resources.

A kit for making seven cDNA libraries from nanogram amounts of total or poly A⁺ RNA. The kit utilizes the proprietary SMART (Switching Mechanism At 5' end of RNA Template) Oligonucleotide to generate high yields of full-length ds cDNA. Control Poly A⁺ RNA, Control Insert and pre-digested λTriplEx2 are included.

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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634901: SMART cDNA Library Construction Kit

A kit for performing ten cDNA syntheses from nanogram amounts of total or poly A+ RNA. The kit utilizes our SMART (Switching Mechanism At 5' end of RNA Template) technology coupled with PCR amplification to generate high yields of full-length double-stranded cDNA suitable for various applications, including Clontech PCR-Select cDNA subtraction, nondirectional cloning, and preparation of complex cDNA probes. The SMARTer PCR cDNA Synthesis Kit includes the same components as the original SMART PCR cDNA Synthesis Kit plus an improved SMARTer II A oligo and SMARTScribe Reverse Transcriptase, which provide higher specificity and increased yield.

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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Comparison of SMARTer Protocols

Comparison of SMARTer Protocols.

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634925: SMARTer PCR cDNA Synthesis Kit

A kit for performing twenty cDNA syntheses from nanogram amounts of total or poly A+ RNA. The kit utilizes our SMART (Switching Mechanism At 5' end of RNA Template) technology coupled with PCR amplification to generate high yields of full-length double-stranded cDNA suitable for various applications, including Clontech PCR-Select cDNA subtraction, nondirectional cloning, and preparation of complex cDNA probes. The SMARTer PCR cDNA Synthesis Kit includes the same components as the original SMART PCR cDNA Synthesis Kit plus an improved SMARTer II A oligo and SMARTScribe Reverse Transcriptase, which provide higher specificity and increased yield.

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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Comparison of SMARTer Protocols

Comparison of SMARTer Protocols.

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SMARTer PCR cDNA Synthesis Kit (Cat. # 634926)

The SMARTer Pico PCR cDNA Synthesis Kit provides a PCR-based method for producing high-quality cDNA from picogram quantities of total RNA. The cornerstone of SMARTer cDNA synthesis is still our unique SMART (Switching Mechanism At the 5' End of RNA Transcript) technology; now with a modified SMARTer II A oligo. This new and improved oligo, combined with SMARTScribe Reverse Transcriptase, provides higher specificity and increased yields. The SMARTer Pico PCR cDNA Synthesis Kit allows you to synthesize high-quality cDNA for array probe generation, cDNA subtraction, "Virtual Northern" blots, or other applications, from as little as 1 ng of total RNA at extremely low concentrations (or in diluted samples). It is especially useful for researchers who have limited starting material, such as RNA derived from laser-capture microscopy samples, cells sorted by flow cytometry, or other extremely small samples.

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SMART(er) cDNA synthesis compared to conventional cDNA synthesis

SMART(er) cDNA synthesis compared to conventional cDNA synthesis. Unlike conventional cDNA synthesis methods, which involve a multiple enzyme/multiple step procedure, the SMART(er) cDNA synthesis protocol is performed by one reverse transcription reaction, in a single tube, with no adaptor ligation or intervening purification steps. Following PCR amplification, SMART(er) cDNA is immediately available for a variety of downstream applications.

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Comparison of SMARTer Protocols

Comparison of SMARTer Protocols.

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634928: SMARTer Pico PCR cDNA Synthesis Kit

The SMARTer RACE 5'/3' Kit allows the synthesis of first-strand cDNA from poly A+ or total RNA via SMART (Switching Mechanism At 5' End of RNA Template) technology, and facilitates the performance of 5'- and 3'-RACE (Rapid Amplification of cDNA Ends) PCR with the kit's Universal Primer Mix. Our carefully designed, specially-modified SMARTer Oligo preferentially captures the 5' ends of the cDNA during cDNA synthesis. Using this SMARTer Oligo, our procedure enriches cDNA pools for 5' sequences, thus increasing the likelihood you will amplify the entire sequence of your gene.

RACE PCR products are amplified with the provided SeqAmp DNA Polymerase, and cloned into the linearized pRACE vector with In-Fusion HD Cloning. The SMARTer RACE 5'/3' Kit has been improved to accommodate larger RNA input volumes and perform better on challenging targets than the original SMARTer RACE cDNA Amplification Kit. The In-Fusion HD Cloning Kit, NucleoSpin Gel and PCR Clean-Up Kit, and Stellar Competent Cells are included for your convenience in cloning RACE products. Gene-specific RACE primers are supplied by the user.

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Overview of the SMARTer RACE 5'/3' Kit workflow

Overview of the SMARTer RACE 5'/3' Kit workflow. Each kit is a complete system, containing the reagents required to recover cloned RACE fragments on the second day.

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Comparing products from the new and old SMARTer RACE kits

Comparing products from the new and old SMARTer RACE kits. Primers were designed using the recommendations in each kit's manual. The samples on this gel image represent a wide range of expression values, and each one has a combined exon length of >10 kb. The new kit is much more successful in amplifying strong, single bands across the sample set.

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634858: SMARTer RACE 5'/3' Kit

The SMARTer RACE 5'/3' Kit allows the synthesis of first-strand cDNA from poly A+ or total RNA via SMART (Switching Mechanism At 5' End of RNA Template) technology, and facilitates the performance of 5'- and 3'-RACE (Rapid Amplification of cDNA Ends) PCR with the kit's Universal Primer Mix. Our carefully designed, specially-modified SMARTer Oligo preferentially captures the 5' ends of the cDNA during cDNA synthesis. Using this SMARTer Oligo, our procedure enriches cDNA pools for 5' sequences, thus increasing the likelihood you will amplify the entire sequence of your gene.

RACE PCR products are amplified with the provided SeqAmp DNA Polymerase, and cloned into the linearized pRACE vector with In-Fusion HD Cloning. The SMARTer RACE 5'/3' Kit has been improved to accommodate larger RNA input volumes and perform better on challenging targets than the original SMARTer RACE cDNA Amplification Kit. The In-Fusion HD Cloning Kit, NucleoSpin Gel and PCR Clean-Up Kit, and Stellar Competent Cells are included for your convenience in cloning RACE products. Gene specific RACE primers are supplied by the user.

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Overview of the SMARTer RACE 5'/3' Kit workflow

Overview of the SMARTer RACE 5'/3' Kit workflow. Each kit is a complete system, containing the reagents required to recover cloned RACE fragments on the second day.

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Comparing products from the new and old SMARTer RACE kits

Comparing products from the new and old SMARTer RACE kits. Primers were designed using the recommendations in each kit's manual. The samples on this gel image represent a wide range of expression values, and each one has a combined exon length of >10 kb. The new kit is much more successful in amplifying strong, single bands across the sample set.

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634859: SMARTer RACE 5'/3' Kit

The Titanium One-Step RT-PCR Kit is designed for the reverse transcription of total or poly A⁺ RNA and the subsequent PCR amplification of a target cDNA in a single reaction tube. The included enzyme mix contains a highly purified Moloney murine leukemia virus (MMLV) reverse transcriptase for first-strand cDNA synthesis, and Titanium Taq DNA Polymerase for PCR amplification. Titanium Taq DNA Polymerase is a blend of a 5'-exonuclease deficient, thermostable DNA polymerase, and TaqStart Antibody for automatic hot start PCR. Reagents for purifying RNA are not supplied.

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Titanium One-Step RT-PCR is more efficient than other commercially available one-step kits

Titanium One-Step RT-PCR is more efficient than other commercially available one-step kits. Reactions were performed in parallel using the Titanium One-Step RT-PCR Kit and two other commercially available one-step kits under the manufacturers' recommended conditions. RT-PCR products were analyzed via agarose/EtBr gel electrophoresis. Human placenta total RNA (1 μg) was used as template for each reaction, and nine different human transcripts were amplified. Lane M: DNA size marker.

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Superior sensitivity of Titanium One-Step RT-PCR

Superior sensitivity of Titanium One-Step RT-PCR. Different amounts of mouse liver total RNA were used as template for TITANIUM One-Step RT-PCR to amplify the beta-actin transcript. The target transcript was reverse-transcribed at 50°C for 1 hr and amplified using 40 PCR cycles; RT-PCR products were analyzed via EtBr/agarose gel electrophoresis. Lane 1: 1 μg of total RNA. Lane 2: 100 ng. Lane 3: 10 ng. Lane 4: 1 ng. Lane 5: 100 pg. Lane 6: 10 pg. Lane 7: 1 pg. Lane 8: no template. Lane M: DNA size markers.

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639503: Titanium One-Step RT-PCR Kit

The Titanium One-Step RT-PCR Kit is designed for the reverse transcription of total or poly A⁺ RNA and the subsequent PCR amplification of a target cDNA in a single reaction tube. The included enzyme mix contains a highly purified Moloney murine leukemia virus (MMLV) reverse transcriptase for first-strand cDNA synthesis, and Titanium Taq DNA Polymerase for PCR amplification. Titanium Taq DNA Polymerase is a blend of a 5'-exonuclease deficient, thermostable DNA polymerase, and TaqStart Antibody for automatic hot start PCR. Reagents for purifying RNA are not supplied.

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Titanium One-Step RT-PCR is more efficient than other commercially available one-step kits

Titanium One-Step RT-PCR is more efficient than other commercially available one-step kits. Reactions were performed in parallel using the Titanium One-Step RT-PCR Kit and two other commercially available one-step kits under the manufacturers' recommended conditions. RT-PCR products were analyzed via agarose/EtBr gel electrophoresis. Human placenta total RNA (1 μg) was used as template for each reaction, and nine different human transcripts were amplified. Lane M: DNA size marker.

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Superior sensitivity of Titanium One-Step RT-PCR

Superior sensitivity of Titanium One-Step RT-PCR. Different amounts of mouse liver total RNA were used as template for TITANIUM One-Step RT-PCR to amplify the beta-actin transcript. The target transcript was reverse-transcribed at 50°C for 1 hr and amplified using 40 PCR cycles; RT-PCR products were analyzed via EtBr/agarose gel electrophoresis. Lane 1: 1 μg of total RNA. Lane 2: 100 ng. Lane 3: 10 ng. Lane 4: 1 ng. Lane 5: 100 pg. Lane 6: 10 pg. Lane 7: 1 pg. Lane 8: no template. Lane M: DNA size markers.

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639504: Titanium One-Step RT-PCR Kit

The Universal Primer Mix (UPM) adds suppression PCR elements to the ends of cDNA generated by our SMARTer RACE 5'/3' Kit (Cat. Nos. 634858 & 634859). The UPM consists of two primers: a long, 45-base primer and a short, 22-base primer. The Long Universal Primer (Long UP) is composed of two parts: the 23 bases at the 3' end of the primer are identical to those at the 5' end of the SMARTer IIA Oligo; the 22 bases at the 5' end of the primer provide the suppression sequence for suppression PCR. The Short Universal Primer (Short UP) sequence is identical to that of the 22 bases at the 5' end of the Long UP. The Short UP, for which the binding site is introduced by the Long UP during the first round of PCR, is present in the UPM at 5 times the concentration of the Long UP.

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634922: Universal Primer Mix