Isolation of Caenorhabditis elegans gene knockouts by PCR screening of chemically mutagenized libraries.
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.
Lesa
Giovanni M
GM
MRC Laboratory for Molecular Cell Biology and Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK. giovanni.lesa@ucl.ac.uk
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000302:isolation & purification; D005020:Ethyl Methanesulfonate / Q000494:pharmacology*; D015698:Genomic Library; D016296:Mutagenesis / Q000187:drug effects* / Q000528:radiation effects; D009153:Mutagens / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
D009153:Mutagens; D005020:Ethyl Methanesulfonate