Universal Riboclone ® cDNA Synthesis ..
Virion RNA served as a template for the RT reaction carried out using the Verso cDNA kit (Thermo, Fisher Scientific, San Jose, CA, USA) or the Maxima Reverse Transcriptase kit (Fermentas). Sequence-specific complementary primers were used in the reaction. The resulting cDNA was amplified in a PCR reaction using Taq polymerase (DreamTaq; Fermentas) or Advantage 2 Polymerase Mix (Clontech-Takara Bio, Madison, WI, USA) and specific primers flanking the PYLCV genes, which are shown in .
(Universal RiboClone cDNA Synthesis ..
cDNA was synthesized in Verso enzyme mix in the presence of PYLCV-specific primers designed to obtain contigs. Second strand synthesis was performed using the Universal RiboClone cDNA Synthesis System (Promega) according to the manufacturer’s instructions. The ds cDNA was purified using a PCR purification kit (Zymo research, Irvine, CA, USA) and the obtained double-stranded fragments were cloned into pUC19 after digestion by SmaI and dephosphorylation (Fermentas). The resulting recombinant plasmids were sequenced to identify viral genomic sequences.
Semi-quantitative reverse transcription-PCR (sqRT-PCR) was carried out on 2 μg of total RNA, by using a two-step RT-PCR kit (Takara, Japan), according to manufacturer’s instructions. Primers were designed from the TDF sequences using a Primer 3.0 web resource. Total RNA was used as the internal RT-PCR standard for checking the quality of the RNA template. PCR conditions were essentially the same as those described earlier for reamplification of TDFs. In order to obtain reproducibility of results, sqRT-PCR was repeated three times.
(Universal RiboClone cDNA Synthesis System, Promega ..
First strand cDNAs synthesis by reverse transcription the products are single stranded. some buffer do not contain DTT, in this case add DTT to 10 mM final. Dec 15, 2006. polymerase RdRp for cDNA synthesis. Dithiothreitol used in the cDNA synthesis step was found to significantly decrease the detection.