Abstract
This protocol details methodologies to generate Caenorhabditis elegans deletion mutants by chemical mutagenesis and to detect them by PCR screening. Approximately, 600,000 worms are grown synchronously, mutagenized with ethyl methane sulfonate, divided in groups of 500 and allowed to self-fertilize for two generations. DNA is prepared from a fraction of each worm population, pooled into a 96-well plate, and screened by PCR with primers positioned 2.5–3.5 kb apart. Cultures containing deletion mutants are subdivided in small worm populations and tested again by PCR to identify positives. Single animals are then cloned from positive cultures, allowed to self-fertilize and identified by PCR genotyping. This method, which takes about a month, gives approximately a 50% chance of finding a deletion of interest larger than 500–600 bp. If a deletion cannot be found, the library can be pooled at lower complexity and screened for smaller deletions using an alternative PCR-based method.
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Change history
29 December 2006
In the version of this article initially published online the background colors for Figure 1 were omitted. This error has been corrected in all versions of the article.
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In the version of this article initially published online the background colors for Figure 1 were omitted. This error has been corrected in all versions of the article.
Acknowledgements
I thank G Schiavo for help in setting up the protocol and G Lalli and N Hopper for suggestions. Work in my laboratory was funded by the Royal Society and by the Medical Research Council. Part of the work described here was funded by Cancer Research UK.
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Supplementary information
Supplementary Data 1
Customizable PCR program for Primary and Secondary PCRs (XLS 24 kb)
Supplementary Data 2
Customizable PCR program for Genotyping PCR (XLS 20 kb)
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Lesa, G. Isolation of Caenorhabditis elegans gene knockouts by PCR screening of chemically mutagenized libraries. Nat Protoc 1, 2231–2240 (2006). https://doi.org/10.1038/nprot.2006.345
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DOI: https://doi.org/10.1038/nprot.2006.345
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