The pCMV-PE2 (Addgene plasmid #132775) and pU6-pegRNA-GG-acceptor (Addgene plasmid #132777) plasmids were a gift from David Liu. pegRNA-encoding plasmids were generated by Golden Gate assembly using a custom acceptor plasmid according to methods described in a previous paper. (23 (link)) pCMV-PE2 was modified to generate a set of plasmids encoding the PE2 variants, which included pCMV-PE2-HF (N497A, R661A, Q695A and Q926A), pCMV-ePE2 (K848A, K1003A and R1060A), pCMV-Evo-PE2 (M495V, Y515N, K526E and R661Q), pCMV-Hypa-PE2 (N692A, M694A, Q695A and H698A) and pCMV-Sniper-PE2 (F539S, M763I and K890N), by incorporating mutations via NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) with gblocks containing mutations (IDT) and site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs).
Protocol detail
Engineered Variants of PE2 Plasmids
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.
Corresponding Organization : Korea University of Science and Technology
Top 5 similar protocols
Variable analysis
independent variables
- PCMV-PE2 variants: pCMV-PE2-HF, pCMV-ePE2, pCMV-Evo-PE2, pCMV-Hypa-PE2, pCMV-Sniper-PE2
- Not explicitly mentioned
- Not explicitly mentioned
- Positive control: None mentioned
- Negative control: None 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!