sgRNA sequences targeting murine G6pdx and non-coding region of DNA (intergenic control) were identified from the mouse GeCKOv2 sgRNA library [36 (link)]. The identified PAM sequences (Supplementary Table 1 ) were subcloned into the pSECC (Addgene, 60820) using the BsmBI restriction endonuclease (NEB R0580S). Virus was produced through PEI (MilliporeSigma, 408727) transfection of vectors and lentiviral packaging plasmids psPax2 and VSVG in 293T cells. Medium containing lentivirus was collected at 48 and 72 h and filtered through a PES filter (0.22 um, MilliporeSigma). Virus was concentrated by ultracentrifugation (~70,000g for 2 h at 4ºC), and the resulting pellet was gently resuspended in an appropriate volume of HBSS (MilliporeSigma, 55037C). Virus was aliquoted and snap-frozen in liquid nitrogen before storing at −80ºC. Viral titering was achieved using Green-Go cells and flow cytometry, as previously described [37 (link)].
Protocol detail
Subcloning Murine G6pdx sgRNA Vectors
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.
293t cells
Bsmbi endonuclease
Cells
Flow cytometry
Frozen
Hbss
Lentivirus
Library
Mouse
Nitrogen
Plasmids
Transfection vectors
Ultracentrifugation
Virus
Corresponding Organization :
Other organizations : Princeton University, Rutgers, The State University of New Jersey, Johnson University
Top 5 similar protocols
Variable analysis
independent variables
- SgRNA sequences targeting murine G6pdx
- Non-coding region of DNA (intergenic control)
- Viral titer
- PEI transfection of vectors and lentiviral packaging plasmids psPax2 and VSVG in 293T cells
- Medium containing lentivirus collected at 48 and 72 h and filtered through a PES filter (0.22 um)
- Virus concentrated by ultracentrifugation (~70,000g for 2 h at 4ºC)
- Virus aliquoted and snap-frozen in liquid nitrogen before storing at −80ºC
- Green-Go cells used for viral titering
- Non-coding region of DNA (intergenic control)
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!