Viruses were generated using plasmid-based reverse genetics (56 (link)– (link)58 (link)). Purified virions were prepared as described previously (16 (link), 59 (link)). To generate mutant viruses, codons for residues S370 and Q371 in the T1L S1 gene plasmid were altered by QuikChange (Stratagene) site-directed mutagenesis. S1 gene sequences were confirmed using the OneStep reverse transcription-PCR (RT-PCR) kit (Qiagen), gene-specific primers, and viral double-stranded RNA (dsRNA) extracted from infected L cells (Trizol, Invitrogen). Genotypes were confirmed by electrophoresis of viral particles (60 (link)). Particle concentrations were determined using the following conversion: 1 absorbance unit at 260 nm (AU260) = 2.1 × 1012 particles. Viral titers were quantified by plaque assay (61 (link)) or fluorescent focus assay (8 (link)).
Onestep reverse transcription pcr rt pcr kit
Manufactured by Qiagen
The OneStep reverse transcription-PCR (RT-PCR) kit is a laboratory equipment product that combines the processes of reverse transcription and polymerase chain reaction (PCR) in a single step. It is designed to facilitate the detection and quantification of RNA targets.
Automatically generated - may contain errors
3 protocols using onestep reverse transcription pcr rt pcr kit
1
Plasmid-based Reverse Genetics for Viral Mutants
2
Viral RNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viral RNA of the generated mutants and reassortants was isolated from 200 μl of infected-cell culture medium by using the High Pure viral RNA kit (Roche). Seg-10 was entirely amplified, and Seg-2 and Seg-6 were partially amplified by using appropriate primers and the One-Step reverse transcription-PCR (RT-PCR) kit (Qiagen). Amplicons were purified by using the Zymoclean gel DNA recovery kit and sequenced according to standard procedures by using the ABI Prism 3130 genetic analyzer (Applied Biosystems), and sequences were verified by using Lasergene SeqMan Pro software (version 11; DNASTAR).
3
Rapid Expression of Recombinant Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Double-stranded cDNAs of the antibody VH and VL genes were produced in the sorted-cells plates using a One-Step reverse transcription PCR (RT-PCR) kit (Qiagen) and then amplified by nested PCR using TransStart Taq DNA polymerase (TransGen Biotech) as previously described.38 (link) The PCR products were sequenced at Sangon Biotech, and the sequences were analyzed using IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest ). Linear expression cassettes were used to quickly express the antibodies. To produce heavy and light chain linear expression cassettes, the cytomegalovirus (CMV) promoter, Ig leader sequence, VH/VL gene, Ig constant region (IgG1), and poly(A) sequence fragment were assembled using overlapping PCR as previously described.39 (link) The full-length antibody sequences were cloned into pcDNA3.4. The plasmids of paired heavy and light chain genes were cotransfected into the Expi293F cells (Thermo Fisher Scientific) according to the manufacturer’s protocol. Antibodies were purified from cell culture supernatants using a HiTrap rProtein A column (Cytiva).
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!