C.elegansGeneKnockoutProtocols.doc

C. elegans Gene Knockout Protocols by Michael Koelle, Valerie Reinke, David Shechner, Keith Kazmer, and Heather Hess Yale University School of Medicine Updated 2/09/03 Credits These protocols are based on methods originally developed in three different groups: 1) Bob Barsteads lab at the Oklahoma Medical Research Center; 2) the NemaPharm Group at Axys Pharmaceuticals; and 3) Ron Plasterks lab at the Netherlands Cancer Institute. Our library construction methods are most similar to those originated at NemaPharm, and include the microtiter culture/frozen worm strategy pioneered there. Rajesh Ranganathan and Peter Reddien in the Horvitz lab developed the special container we use to freeze worms in microtiter plates. Our library screening protocols are based on the poison primer method pioneered in the Barstead and Moerman groups. We have varied most aspects of the procedure to improve efficiency and success rate. Thus our methods differ from those of others in a number of details. Speed and success rate of the method This procedure requires an initial investment of about 2 weeks of part-time work to pilot the methods followed by ~3 weeks of full-time work (if two individuals work together) to construct a frozen mutant library. The library can be stored indefinitely and can be screened at least 200 times. Once the library is constructed one can isolate a live mutant in a gene of interest in only 2-3 weeks of work. Using these methods we have so far succeeded in obtaining one to three mutant alleles for almost every gene we have worked on. We believe this technology is now successful enough that it is worth the investment of time and effort for any small C. elegans laboratory. These protocols are written such that there are quantitative quality control assessments at each stage of library construction. Thus the success of each part of the procedure can be verified along the way, and any unsuccessful batches can be discarded immediately if a failure occur