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Bioengineering

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JoVE Journal Bioengineering
Opførelse af CRISPR Plasmids og påvisning af Knockout Effektivitet i pattedyrceller gennem en Dobbelt Luciferase Reporter System
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Opførelse af CRISPR Plasmids og påvisning af Knockout Effektivitet i pattedyrceller gennem en Dobbelt Luciferase Reporter System

Article DOI: 10.3791/59639-v • 05:51 min • December 5th, 2020
December 5th, 2020 •
*1, *1, *1, 1, 1, 2, 1
1Qingdao Agricultural University, Qingdao, China, 2University of Cantabria, Santander y Torrelavega, Spain
* These authors contributed equally

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Her præsenterer vi en protokol, der beskriver en strømlinet metode til effektiv generering af plasmider, der udtrykker både CRISPR-enzymet og tilhørende enkelt guide RNA (sgRNAs). Co-transfektion af pattedyrceller med denne sgRNA/ CRISPR vektor og en dobbelt luciferase reporter vektor, der undersøger dobbelt-streng pause reparation giver mulighed for evaluering af knockout effektivitet.

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Bioengineering CRISPR single guide RNA (sgRNA) Genredigering Dual luciferase reporter system enkelt streng glødning PCR-baseret detektion
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