DRR-pvT1 was amplified by PCR using primers DR1R and U100Fw2 in the first round (94°C, 1.5 min; 25 cycles 94°C 15 s, 62°C 30 s, 68°C 10 min; 68°C 2 min) and primers DR421R and TJ1F in the second round (94°C, 1.5 min; 25 cycles 94°C 15 s, 64°C 30 s, 68°C 1.5 min; 68°C 2 min). DRL-pvT1 was amplified by STELA (see below) followed by a secondary PCR using primers DR421R and TJ1F. The short pvT1 amplicons were size-separated by electrophoresis in a 3% NuSieve (Lonza) agarose gel, extracted, and Sanger sequenced using primer TJ1F. The number and pattern of (TTAGGG) and degenerate telomere-like repeats were identified and color-coded manually to generate pvT1 repeat patterns. For comparative analysis, the DRR-pvT1 repeat patterns were aligned manually by dividing them into the proximal, central, and distal regions with respect to the DR421 primer and distinctive motifs identified (Supplementary file 3 ).
Nusieve
Manufactured by Lonza
Sourced in Australia, Japan
NuSieve is a laboratory equipment product designed for agarose gel electrophoresis. It is used for the separation and analysis of DNA or RNA molecules. The core function of NuSieve is to provide a reliable and consistent platform for the electrophoretic separation of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Gelred, by Biotium (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Avaii, by New England Biolabs (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
5 protocols using nusieve
1
Characterization of Telomeric Repeats in pvT1
2
Gel Electrophoresis of PCR Products
3
Quantifying Gene Expression via RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One to two micrograms of total RNA was subjected DNase treatment using one unit of RQ1 RNase-free DNase followed by cDNA synthesis using both 0.2 µM forward and reverse primers in a 50 µL reaction containing (1X PCR buffer, 0.2 mM dNTP’s, 1.5 mM MgCl2, 0.2 µM primer(s), 12 U RNase out (Invitrogen), 60 U SuperScript III (Invitrogen) and 1.5 unit Platinum Taq DNA Polymerase). Following cDNA synthesis, the reaction was then subjected to PCR for 35 cycles. PCR products were visualized on a 3% NuSieve (Lonza, Victoria, Australia)/1X TBE gel.
4
RFLP Analysis of Exon 4 Region
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The first round of the two-step nested PCR protocol for RFLP amplified the exon 4 region (PRJEB506 position: 31,521,758–31,522,025 bp) from single worm lysates as described in 2.5.1 except using primers Hco-Intron4-F and Hco-Exon4-R. A 1 μl aliquot of the PCR product from round 1 was diluted 1:20 and added to 19 μl GoTaq G2 Flexi PCR reaction mix using primers Hco-Exon4-F and Hco-Exon4-R and amplified as follows: initial denaturation 95 °C for 2 min, followed by 40 cycles (denaturation 95 °C for 30 s, annealing TAoC for 30 s, and extension 72 °C for 15 s) with the final extension at 72 °C for 5 min 20 μl of nested PCR product was digested using AvaII (NEB, R0153S) for 7–12 h at 37 °C in CutSmart buffer (NEB, B7204S). Digested products were then visualised on a 4% NuSieve (Lonza, 50090) agarose gel with SafeView Nucleic Acid Stain.
5
Quantification of TiLV by RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TiLV was quantified using SYBR® green-based RT-qPCR assay, according to Tattiyapong et al. (2018) (link). Briefly, the 10 L reaction was comprised of 4 L of cDNA, 5 L of 2× iTaq™ universal SYBR® green-based supermix (Bio-Rad Laboratories; Hercules, CA, USA), 0.3 L each of 10 M forward and reverse primers (accession no. KJ605629), and then the mixture was adjusted to a volume of 10 L using DNase free water. All samples were run in triplicate. Later, the reaction was performed in a real-time PCR thermocycler (CFX96 Touch™; Bio-Rad Laboratories; Hercules, CA, USA) with 2-step cycle conditions including a cycle of 95 °C for 3 min, and 40 cycles of 95 °C for 10 s and 60 °C for 30 s. To determine the specificity of qPCR reactions, the products were further verified by melting curve analysis with the step of 65-95 °C with 5 °C per 5 s increment, and products were separated on low melting temperature agarose gel (NuSieve®, Lonza, Japan) and visualized under UV light. The amount of virus was then extrapolated from the Ct value of each sample by comparing it to the standard curve as previously described (Nicholson et al., 2018) .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!