Transcriptor High Fidelity Cdna Synthesis Kit Pdf Download ##BEST##

PrimeScript Reverse Transcriptase (PrimeScript RT), included in the PrimeScript High Fidelity RT-PCR Kit, efficiently synthesizes cDNA thanks to its exceptionally strong strand-displacement activity. PrimeScript RT is a modified, recombinant RNase H Minus MMLV (Moloney Murine Leukemia Virus) reverse transcriptase that is robust, versatile, and well-suited for applications requiring full-length cDNA, such as RT-PCR gene expression studies. PrimeScript RT can be used for reverse transcription of virtually any RNA template, including templates that are GC-rich and/or have high levels of secondary structure. Because of the excellent extension capability of this enzyme, cDNA synthesis can be performed at 42C, decreasing the risk of RNA degradation that often occurs during conventional reactions performed at higher temperatures.

During RT-PCR gene expression studies, accurate reverse transcription and PCR amplification are essential. The PrimeScript High Fidelity RT-PCR Kit includes all of the reagents needed for high-fidelity amplification of cDNA from total RNA or mRNA, and is designed for two-step endpoint RT-PCR. This kit includes PrimeScript High Fidelity Reverse Transcriptase, a highly accurate MMLV reverse transcriptase, and PrimeSTAR Max DNA Polymerase, which has the highest fidelity of any commercially available PCR enzyme. PrimeSTAR Max DNA Polymerase is formulated as a premix and offers high amplification efficiency as well as compatibility with a wide range of template concentrations. As a result, this kit is well-suited for any cDNA studies requiring high fidelity, such as RT-PCR gene expression studies (including gene-splicing studies where alternative splicing products must be distinguished, or when discerning between closely related gene family members), cDNA library construction, and transcriptome analysis.

Transcriptor High Fidelity Cdna Synthesis Kit Pdf Download

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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.

Previous approaches to detect sRNAs on ONT devices involve using chemical modifications to add bulky residues to slow the voltage driven translocation of molecules through the pore and increase the signal, however it is not possible to achieve whole transcriptome sequencing using this method [19]. In another approach, it has been shown that it is possible to sequence small DNA molecules using circularization of padlock probes followed by rolling circle DNA amplification [20], however this method required a specific probe for each target and has not been demonstrated on RNA targets. Rolling circle amplification allows for high fidelity sequencing because the original molecule is copied multiple times in tandem repeats and these repeats can be used to assemble a high accuracy consensus sequence from noisy data. Rolling circle amplification has also been used as a strategy to increase the accuracy of nanopore sequencing of long RNA, using reverse transcription followed by circularization of the cDNA product [21].

All previous approaches using rolling circle amplification to aide in nanopore sequencing have used phi29 DNA polymerase due to its high processivity and strong strand displacement. We reasoned that a highly strand displacing reverse transcriptase (RT) could achieve the same goal directly on RNA templates and would therefore allow for direct amplification of sRNA. One candidate group of enzymes to perform such a function are Group II intron reverse transcriptases. Group II introns are retrotransposons that encode a self-splicing ribozyme as well as a reverse transcriptase. To complete the retrotransposition cycle, the intron-encoded reverse transcriptase must accurately copy its cognate ribozyme which is large and highly structured. While homologous to the retroviral encoded reverse transcriptases typically used in biotechnology, this selection pressure has led to the evolution of high processivity, high fidelity and strong strand displacement activity in the Group II intron RTs [22].

In this paper we show that it is possible to perform rolling circle reverse transcription using Group II intron RTs, with much better performance than retroviral RTs. We developed an efficient sequencing strategy that allows for adapter ligation, circularization and rolling circle reverse transcription of cellular sRNAs without intermediate purifications. The resulting cDNA products can be used directly or further amplified before being sequenced on the ONT MinION device. Finally, we show that our method for converting sRNAs into concatemeric cDNAs (SR-Cat-Seq) can be used to reconstruct high-fidelity and low-bias sRNA transcriptomes from error-prone nanopore data.

Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.

Hi Aykut,

Do you know if your gene is expressed at the stage from which you have total RNA from? In general, you want to extract total RNA at a stage when the gene you want to clone is expressed at high levels.

I always found oligo dT priming better for making cDNA (especially for longer constructs). The transcriptor high fidelity cDNA kit from Roche is especially good for this (but any cDNA synthesis kit should work). Make sure you have good quality total RNA from the right developmental stage.

In general, shorter primers would give less specificity, not more. Since you had a non-specific product with a longer primer pair, you are not likely to get specific PCR product with a shorter primer pair.

The efficiency of m1 incorporation during in vitro transcription was investigated in four different RNA substrates of varying length and sequence. The base composition of the synthesized RNA was analyzed with ultra-high performance liquid chromatography coupled with mass spectrometry. For the long synthetic RNAs with length ranging from 1122 to 4178 nucleotides, the integrity of the RNA was determined using Bioanalyzer. Synthesis of full-length RNAs of expected sizes were observed in reactions performed in the presence of m1 with both T7 RNAP and SP6 RNAP (Supplementary Fig. 1a). Furthermore, similar to , total m1 TP incorporation was as expected in RNA sequences RNA1 (a 1122 nucleotide synthetic sequence that includes all possible four-base combinations)27 and Cypridina luciferase mRNA sequence (CLuc mRNA) when in vitro transcription was performed with T7 RNAP (Supplementary Fig. 1b,c). In order to determine that the RNA synthesized in presence of m1 m1TP is indeed of expected length, we subjected two short RNAs, 30-nucleotide RNA and 60-nucleotide long, to intact mass spectrometry analyses (Supplementary Fig. 2). Irrespective of which uridine analog was present in the reaction, the mass of the predominant species observed in reactions performed with T7 RNAP correspond to the run-off transcripts and few non-templated additions were observed28 suggesting that similar to and uridine, m1 is incorporated efficiently during in vitro transcription and the modifications did not disrupt the synthesis of full-length run-off products.

For synthetic mRNA-based applications, high yield of RNA from the in vitro transcription reaction is desirable and reactions are typically performed with high concentrations of rNTPs. The recommended high-yield rNTP concentrations are different for T7 RNAP (40 mM rNTP) and SP6 RNAP (20 mM rNTP). In order to ensure that the differences in combined error observed for T7 RNAP and SP6 RNAP are not due to differences in the rNTP concentrations in the reactions, we performed in vitro transcription reactions with T7 RNAP under low rNTP reaction conditions with either 20 mM or 10 mM rNTP (total). The total combined error rates as well as the base substitution errors observed in unmodified RNA1/RNA2 when reactions were performed with T7 RNAP under low rNTP (10 or 20 mM) reaction conditions were comparable to that observed with high rNTP (40 mM) reaction conditions (Supplementary Table 6) suggesting that the overall rNTP concentration in the reaction does not affect fidelity of uridine incorporation and the differences in error rates observed with T7 and SP6 RNAP are not due to differences in the reaction conditions. be457b7860