DNA mixtures were micro-injected into the gonads of young adult worms. Plasmids for injection were prepared using a midiprep plasmid purification kit (Qiagen, no. 12143). For Co-CRISPR, we injected 50 ng/µl each vectors [Cas9 vector, unc-22 sgRNA vector (Co-CRISPR), two untested-sgRNAs, and pRF4::rol-6 (su1006 )] (Figure 2A ). Micro-injection mixtures for HR contained 50 ng/μl each Cas9 vector, sgRNA vector, pRF4::rol-6 (su1006 ), and HR donor vector. The final concentration of DNA in the injection mix did not exceed 200 ng/µl. For injection mixes with five different plasmids, 40 ng/µl of each plasmid was added. For HR experiments, we injected 40–60 worms and for disruptions, 20–30 worms. After recovering from injection, each worm was placed onto an individual plate.
Protocol detail
CRISPR-Cas9 Genome Editing in C. elegans
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 : Howard Hughes Medical Institute
Top 5 similar protocols
Protocol cited in 35 other protocols
Variable analysis
independent variables
- Plasmids for injection
- Concentration of DNA in the injection mix
- Phenotypic outcomes of the injected worms
- Micro-injection of DNA mixtures into the gonads of young adult worms
- Use of a midiprep plasmid purification kit (Qiagen, no. 12143) to prepare plasmids for injection
- Injection of 50 ng/µl each Cas9 vector,
unc-22 sgRNA vector (Co-CRISPR), two untested-sgRNAs, androl-6 (su1006 ) for Co-CRISPR experiments - Injection of 50 ng/μl each Cas9 vector, sgRNA vector,
rol-6 (su1006 ), and HR donor vector for HR experiments - Injection of 40 ng/µl of each plasmid for injection mixes with five different plasmids
- Injection of 40–60 worms for HR experiments and 20–30 worms for disruptions
- Placing each injected worm onto an individual plate after recovering from injection
- Positive control: None specified
- Negative control: None specified
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!