Target genome fragments of different GoAstV genotypes were amplified using primers designed in this study. The PCR products were then cloned into the pGEM-T easy Vector system (Promega, Madison, WI) following the instruction. The recombinant vectors were then transformed into E. coli DH5α competent cells (CWBIO, Beijing, China), which was confirmed by sequencing in Shanghai Shengong Life Technology (Shanghai, China). Recombinant plasmids were linearized by EcoRI (Promega, Madison, WI). The linear DNA was precipitated by phenol: chloroform: isoamyl alcohol, and then suspended in water before in vitro transcription reactions using Riboprobe in vitro Transcription System with T7 RNA Polymerase (Promega, Madison, WI) following the manufacturer's instructions. The in vitro transcribed viral RNA was obtained and the DNA template with RQ1 DNase was removed at the last step to eliminate the DNA's effect or interference with the sensitivity tests. The transcribed RNA concentration was quantitated by the DeNovix DS-11 Spectrophotometer and calculated in copy numbers.
Riboprobe in vitro transcription system with t7 rna polymerase
Manufactured by Promega
The Riboprobe in vitro Transcription System with T7 RNA Polymerase is a laboratory equipment used for the in vitro synthesis of RNA. It contains the T7 RNA polymerase enzyme, which is responsible for the transcription of DNA into RNA.
Automatically generated - may contain errors
2 protocols using riboprobe in vitro transcription system with t7 rna polymerase
1
Preparation of Goose Astrovirus RNA
2
In vitro Transcription of TMUV RdRP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro-transcribed NS5 RdRP domain RNA of a TMUV field strain isolated from Peking duck (TMUV-SD2010)4 (link) was used to determine the detection limit of the assay. The NS5 RdRP domain (1084 bp) of TMUV-SD2010 was amplified using published procedures46 (link) and cloned into the pGEM-T easy Vector system (Promega, Madison, WI, USA) as described in our previous study4 (link). The recombinant plasmid pGEM-NS5 was linearized by EcoRI (Promega), extracted using phenol–chloroform–isoamyl alcohol (25:24:1), precipitated using ethanol, and suspended in water before using the DNA for in vitro transcription reactions. The production of “run-off” transcripts derived from the inserted M1 segment used the Riboprobe®in vitro Transcription System with T7 RNA Polymerase (Promega) following the manufacturer’s instructions. The transcribed RNA concentration was quantitated using a NanoDrop™ 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and copy numbers were calculated as previously described47 (link) before making a 10-fold serial dilution for sensitivity testing and generating the standard curve.
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!