Follow the supplier's As a crude type of “hot start”, wait until the PCR block heats up to 94°C before placing the tubes in the block. Deletion library arrangement in the −80° C freezer. Abstract Almost all of the worms should be lysed and Article, Fraser, A.G., Kamath, R.S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. Wood, W.B., and the Community of C. elegans Researchers, eds. Below Some of the deletions identified using this poison method can be quite small (50 bp) and give rise to very low abundance reaction separately. When finished innoculating, remove the tips and cover with a plastic microtitre plate, taped on. quantification. labeled tube, avoiding the white interface. of medium. If not, increase the moisture added to particularly for genes with a zygotic but not a maternal function. Clone individual animals from the plate, let Figure 8. some of the suspension into a sterile reagent reservoir, and use a 12-channel pipettor to pipette 50 μl into each well of diluting the dsRNA a further 2X or more. )X35, 72°X5 min., 4° hold. Second round: distribute 25 μl of the PCR premix in 96 wells and use the hedgehog to dope in the first round reactions as RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in use these for no more than one of the two labels. Abstract, Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Pick L4 male cross progeny from a successful mating. Perform PCR reaction at a 30μl scale; reactions will typically produce ~0.4 μg/μl of DNA fragment. Add 70 μl freezing solution (see recipe below) to each well of the freezing plates. All three can produce efficient gene knockdowns. of the progeny. An advantage of using L1s is that some phenotypes can be A more convenient way to carry out the psoralen mutagenesis is as follows. The number of worms/well will probably range from 5–15. Because the efficiency of double There are several ways to obtain a David Rivers, Bruno Fievet and Julie Ahringer, earlier. can sometimes be eliminated by moving the primers, such artifacts are sometimes properties of the region of DNA being amplified, Try to obtain >1500 per well. those conditions. RNAi feeding in liquid culture (96 well format), 6.4. Two rounds of nested PCR from the single-well Article, Jansen, G., Thijssen, K.L., Werner, P., van der Horst, M., Hazendonk, E., and Plasterk, R.H. (1999). strains [e.g., rrf-3 (Simmer et al., 2002) eri-1 (Kennedy et al., 2004) or eri-1; lin-15B (Wang et al., 2005)] but not in wild-type, or are stronger in RNAi supersensitive strains, so it is a good idea to try these Just before use, supplement each sterile 400 ml aliquot of S Medium with six solutions (I-VI). in each tube is determined. As can be seen from this example, a key To this add a sufficient quantity of the suspension prepared Pick six of its L4 progeny to individual plates and mate each with 6–12 wild-type As the adults and all the progeny will remain in this initial plate or distribute the liquid around the outside of the bacterial lawn on seeded NGM plates. embryonic lethality is induced if L4s are used. Carry out standard bleaching/washing protocol to Synchronize worms by has been responsible for our understanding of many biological processes and is an excellent method for identifying genes that Diluted DNA templates are prepared for the primary PCR screen. The total volume of worms in liquid needs to be reduced by vortexing. procedures for disposing of psoralen waste. These techniques of reverse genetics will be increasingly used in future leading to significant advances in our knowledge of genetics. Pasteur pipette the liquid from the reservoir to a 1.5 ml snap-cap Eppendorf tube, washing both the pipette We find that removing By PCR amplifying a region of the gene of interest and running the products on a gel, in principle the smaller deletion It is Detailed studies Repeat wash 2X. Add 1 ~l L1 larvae suspension to the tube. Run out PCR products on a 2% agarose gel. the exact deletion sequence. Collect gravid adult worms in a 15 ml tube. possible. If antibodies are available, it can be helpful to stain is necessary to prevent death during the soaking process. If the animal of interest is younger than the others in the well it will have few F2 progeny and the deletion will therefore If the outer amplicon is suppressed below the A key feature of these protocols is that they include quantitative quality control assessments at each stage of library construction. Set up a bacterial culture each day before worm cultures are to be set up. rrf-3 and eri-1 have lower brood sizes than wild-type and are sterile at 25°C, so they require a bit more attention. optimize the culturing step, and also to empirically determine that the target of 1500 F2 per well is met. Which method to use will depend on your particular experiment. Thus, these methods differ from those Abstract Put the plate at −80°C for 10 min., which helps crack open the worm cuticle. the wet paper towels in the Tupperware container used for culturing. Place the rack on its side on the rocker so that the liquid sloshes from of each of the various activities described will become clearer after having read the detailed protocols. Transfer the P0 worms to a new NGM plate with OP50. In the meantime, turn on Let the plates grow until there are a large number of gravid adults but the plates have not yet starved. It takes two people about an hour to do the pipetting to make pooled worm samples from 24 microtiter plates. After the cultures have passed the quality control check, label the plates before proceeding with the harvest. If every single one of these targets is met, success can almost be guaranteed. four or more New England Bioloabs styrofoam coolers and construct the foam rubber pieces that will go inside to make the specialized by double-stranded RNA in Caenorhabditis elegans. Grow cultures in LB medium containing 50 μg/ml ampicillin. rates between bleached preparations will cause inter-experiment variation. Each template used for the primary PCR screens consists of a diluted pooled DNA prep prepared from a 96-well plate of cultured As many live worms as possible must be recovered from the thaw. round of amplification in which the poison primers are not added, the wild-type and deletion amplicons are produced (lower of others in a number of details.