All required enzymes, optimized buffers, RNase free water and high quality dNTPs are supplied along with the product package.
Figure 1: NG dART RT-PCR Kit: Temperature gradient RT-PCR from purified RNA. RNA was isolated from swine liver (Sus scrofa). mRNA for Sus scrofa arginase I was reverse transcribed and amplified with NG dART Reverse Transcriptase. PCR amplification with OptiTaq DNA Polymerase [3.5 µl]. (Cat. No. E2600). Amplicon length: 1137 bp. RT-PCR products were analyzed on an 1% [w/v] agarose gel.
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. Obviously, no PCR-detectable traces of contaminating DNA remained after purification.
Reverse transcription was performed using the NG dART reverse transcriptase. 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.
2 µl cDNA were taken as template DNA for subsequent PCR amplification (30 cycles) using OptiTaq DNA Polymerase (included in the NG dART RT-PCR Kit) 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.
Following PCR, 20 µl were applied to an agarose gel and analyzed in parallel with 3.5 µl of PerfectPlus 1 kb Ladder.
Predicted amplicon size: 1133 bp. PCR amplicon covers ~80% of the complete Sus scrofa ArgI mRNA (1401 nt, GenBank accession number NM_214048).
NG dART Reverse Transcription Kit - Package Contents