dART 2-Step RT-PCR Kit
cDNA synthesis kit optimized for sensitive and highly specific RT. Dedicated for rare RNA templates and for quantitative analyses.
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Figure 1: dART RT Kit + OptiTaq DNA Polymerase: RT-PCR from purified RNA. RNA was isolated from swine liver (Sus scrofa). RT-PCR products were analyzed on an 1% [w/v] agarose gel.
- Lane MW: Molecular weight marker: PerfectPlus 1 kb Ladder [3.5 µl]. MW band sizes [kb] are displayed at the left border, MW mass per band [ng per 3.5 µl gel loading] are displayed at the right border of the gel picture, respectively.
- Lane RT-: PCR from purified, non-DNase digested RNA did not yield any PCR product, confirming the absence of contaminating DNA from purified RNA.
- Lane RT+: PCR from cDNA (derived from the RNA sample shown in lane 2), specific for the Sus scrofa type I arginase (liver-type arginase) as described below, gave an amplicon of the expected size (~1.1 kb).
RNA was isolated from pork liver using the GeneMatrix Universal RNA Kit (RT-PCR results shown) and from swine blood using the GeneMatrix Human Blood RNA Kit (RT-PCR results not displayed; fainter RT-PCR product obtained due to generally low RNA amounts in blood samples). According to the kit supplied protocols, DNase digestion was not performed. Purified RNA was used as negative control template during RT-PCR (lane RT-). Obviously, no PCR-detectable traces of contaminating DNA remained after purification.
Reverse transcription was performed using the dART RT Kit. Priming was initiated with the gene specific (reverse) primer 5'-TCA CAC CAA GAG GGA ATT GAT AC-3'. RT was performed for 20 min at 50°C (no optional initial heating and subsequent annealing steps were performed prior to RT reaction; an optional RNase H digestion step following RT, as decribed in the product manual, was not conducted as well).
2 µl cDNA were taken as template DNA for subsequent PCR amplification (30 cycles) using OptiTaq DNA Polymerase and the primer pair 5'-CGA TGA GTT TCA AGT CAC AAT CC-3' (forward) / 5'-TCA CAC CAA GAG GGA ATT GAT AC-3' (reverse) in a total PCR reaction volume of 25 µl. Predicted amplicon size: 1133 bp. PCR amplicon covers ~80% of the complete Sus scrofa ArgI mRNA (1401 nt, GenBank accession number NM_214048).
Following PCR, 20 µl were applied to an agarose gel and analyzed in parallel with 3.5 µl of PerfectPlus 1 kb Ladder.
dART RT-PCR Kit - Package Contents
Reverse Transcription / cDNA Synthesis
- dART Reverse Transcriptase
- Ribonuclease Inhibitor
- 5x cDNA Buffer
- dNTP mix 5 mM each
- DTT [100 mM]
- RNase-free water
- Random Hexamers
- RNase H (E. coli)
- dART Reverse Transcription Kit, no thermostable DNA Polymerase included
- dART 1-Step RT-PCR Kit, with OptiTaq DNA Polymerase and RNase Inhibitor
- RNA Extraction & Purification Kits
- SG qPCR Master Mixes (2x) for qPCR / RealTime PCR
- EURx PCR / DNA CleanUp Kit for purification of PCR products