11 genes that showed evidence of mutations in the T7 Endonuclease I assay were chosen for independent confirmation via Sanger sequencing. PCR products corresponding to these 11 sites were cloned into PCR-4-Blunt-Topo using a Topo cloning kit (Life Technologies) and transformed into XL-1 Blue cells. Plasmid DNA was isolated for multiple colonies from each transformation and then sent for DNA sequencing using either the T7 (5′-TAATACGACTCACTATAGGG-3′) and T3 (5′-ATTAACCCTCACTAAAGGGA-3′) primers or the M13F (5′-GTAAAACGACGGCCAG-3′) and T3 primers.
Protocol detail
Sanger Sequencing Validation of Genetic Mutations
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 :
Other organizations : Center for Cancer Research, Massachusetts General Hospital, Harvard University
Top 5 similar protocols
Protocol cited in 55 other protocols
Variable analysis
independent variables
- Genes that showed evidence of mutations in the T7 Endonuclease I assay
- Confirmation of mutations via Sanger sequencing
- PCR products corresponding to the 11 sites were cloned into PCR-4-Blunt-Topo using a Topo cloning kit (Life Technologies) and transformed into XL-1 Blue cells
- Plasmid DNA was isolated for multiple colonies from each transformation and then sent for DNA sequencing using either the T7 and T3 primers or the M13F and T3 primers
- Positive control: Not explicitly mentioned
- Negative control: Not 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!