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AccuScript High-Fidelity 1st Strand cDNA Synthesis Kit

RNA from subconfluent cultures of NFs and AFs wasisolated using an RNeasy micro kit (Qiagen, Gaithersburg, MD, USA).DNase I (Qiagen) was applied in RNA solution to properly removegenomic DNA, and the purification procedure was repeated. Reversetranscription was performed with 1 g of total RNA and anAccuScript High Fidelity First Strand cDNA Synthesis kit(Stratagene, San Diego, CA, USA) according to the manufacturer'sinstructions. For negative control, the same reverse transcriptionreaction without reverse transcriptase was performed. The PCRreaction was performed with REDTaq ReadyMix (Sigma-Aldrich). Allprimers are listed in .

AccuScript high fidelity reverse transcriptase accurately synthesizes cDNA

Nucleotide distribution surrounding fragment ends and calculation of bias weights. (a) Sequence logos showing the distribution of nucleotides in a 23 bp window surrounding the ends of fragments from an experiment primed with 'not not so random' (NNSR) hexamers []. The 3' end sequences are complemented (but not reversed) to show the sequence of the primer during first-strand synthesis (see Figure 1). The offset is calculated so that zero is the 'first' base of the end sequence and only non-negative values are internal to the fragment. Counts were taken only from transcripts mapping to single-isoform genes. (b) Sequence logo showing normalized nucleotide frequencies after reweighting by initial (not bias corrected) FPKM in order to account for differences in abundance. (c) The background distribution for the yeast transcriptome, assuming uniform expression of all single-isoform genes. The difference in 5' and 3' distributions are due to the ends being primed from opposite strands. Comparing (c) to (a) and (b) shows that while the bias is confounded with expression in (a), the abundance normalization reveals the true bias to extend from 5 bp upstream to 5 bp downstream of the fragment end. Taking the ratio of the normalized nucleotide frequencies (b) to the background (c) for the NNSR dataset gives bias weights (d), which further reveal that the bias is partially due to selection for upstream sequences similar to the strand tags, namely TCCGATCTCT in first-strand synthesis (which selects the 5' end) and TCCGATCTGA in second-strand synthesis (which selects the 3' end). Although the weights here are based on independent frequencies, we found correlations among sites in the window and take these into account in our full model to produce more informative weights (see Supplementary methods in Additional file ). A similar figure to this for the standard Illumina Random Hexamer protocol and plots similar to (d) for all datasets in the paper can be found in Figures S1 and S2 of Additional file respectively.

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The bias weight b(t, i, j) factors as where i and j are the 5' and 3' endpoints, respectively, of a fragment mapped to transcript t. The and weights measure sequence-specific bias and are found by calculating the ratio of the probability of the sequence surrounding the fragment end under the biased model to the uniform (null) model. Note that we model both ends separately due to the differences in sequence selectivity between the priming steps during first- and second-strand synthesis. In our method, these probabilities are actually learned from the data using a variable length Markov model [] to capture dependencies between positions in the sequence. Complete details are in the Supplementary methods in Additional file .

The cDNA was synthesized using oligo-(dT) primer and AccuScript™ high fidelity 1ST strand cDNA synthesis kit (Stratagene) at 65℃ for 5 minutes, at 42℃ for 1 hour and at 70℃ for 15 minutes.

high fidelity first-strand cDNA synthesis kit ..

cDNA was synthesized from 2 μg total RNA using the Maxima First Strand cDNA Synthesis Kit ..

The cycle threshold values were converted into relative expression levels using standard curves generated for the mouse tapasin and mouse GAPDH primers. The relative expression levels obtained from four tapasin and four glyceraldehyde 3-phosphate dehydrogenase (GAPDH) qRT-PCR analyses of a cDNA preparation were averaged. Next, the relative expression of tapasin was normalized to the relative expression of GAPDH for each line. The normalized relative expression of mouse tapasin in R1E cells was set as the control and used to calculate the change in mouse tapasin mRNA expression in the R1.1, R1E-Db, and R1E-Db2m cells. According to the results of an F-test, the two-sample equal variance Student's t-test was used to determine the significance of the difference in mouse tapasin mRNA expression in R1E versus R1.1 cells. Results with R1E were compared to results with either R1E-Db or R1E-Db-β2m by the two-sample unequal variance Student's t-test.

Overview of a typical RNA-Seq experiment. RNA is initially fragmented (1) followed by first-strand synthesis priming (2), which selects the 3' fragment end (in transcript orientation), to make single stranded cDNA. Double stranded cDNA created during second-strand synthesis (3), which selects the 5' fragment end, is then size selected (4) resulting in fragments suitable for sequencing (5). Sequenced reads are mapped to opposite strands of the genome (6), and in the case of known transcript or fragment strandedness, the read alignments reveal the 5' and 3' ends of the sequenced fragment (see Supplementary methods in Additional file ). All arrows are directed 5' to 3' in transcript orientation.

High Fidelity First Strand cDNA Synthesis Kit, ..
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  • AccuScript High Fidelity 1st Strand cDNA Synthesis kit ..

    Approximately 200 ng of RNA was used with the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit ..

  • AccuScript High Fidelity 1st Strand cDNA Synthesis Kit ..

    cDNA synthesis kit

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    (AccuScript High Fidelity 1st Strand cDNA Synthesis Kit ..

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