Site-directed mutagenesis was performed to introduce mutations into the exact copy of BTV-1 S5(VP5) or S7 (VP7) (pUC19BTV1T7S5 or pUC19BTV1T7S7) complementary DNA plasmid for virus recovery assay, as previously described14 (link),20 (link),29 (link). Briefly, BTV RNA transcripts were generated from the digested cDNA plasmid clones using a mMACHINE T7 transcription kit (Thermo Fisher). A monolayer of BSR cell (BHK-21 subclone) in six-well plates at 100% confluence was transfected with a mixture of ten different BTV RNA transcripts using Lipofectamine 2000 reagent. At 4 h posttransfection, the medium was replaced with a 6-ml overlay consisting of minimal essential medium, 2% FBS and 1.5% (w/v) agarose. Then the plates were incubated at 35 °C in 5% CO2 for 72 h to allow plaques to appear.
Protocol detail
Recovering Mutant Bluetongue Viruses
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Agarose
Assay
Cdna
Cell
Clones
Lipofectamine 2000
Mutations
Plaques
Plasmid
Site directed mutagenesis
Transcription
Virus
Corresponding Organization :
Other organizations : University of California, Los Angeles, London School of Hygiene & Tropical Medicine, University of London, California NanoSystems Institute
Top 5 similar protocols
Variable analysis
independent variables
- Site-directed mutagenesis to introduce mutations into the exact copy of BTV-1 S5(VP5) or S7 (VP7) (pUC19BTV1T7S5 or pUC19BTV1T7S7) complementary DNA plasmid
- Virus recovery assay
- Monolayer of BSR cell (BHK-21 subclone) in six-well plates at 100% confluence
- Mixture of ten different BTV RNA transcripts
- Lipofectamine 2000 reagent
- Minimal essential medium with 2% FBS and 1.5% (w/v) agarose
- Incubation at 35 °C in 5% CO2 for 72 h
- No positive or negative controls were explicitly mentioned.
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!