PrimeScript 1st strand cDNA Synthesis Kit
PrimeScript 1st strand cDNA Synthesis Kit
Brand: Takara Bio.
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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.
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)
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 |
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.
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.
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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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.
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.
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.
