hi fellows: I'm now trying to do rescue experiment by using PCR product, bu I can hardly get a line? could someone give me some suggestions what aspects should I take care of. like the concentration, and because this is a new gene, I don't know exactly how long the promotor is
Have you successfully made transgenics in C. elegans before and are simply having trouble with this new gene? Or are you new to the technique? In either case, I would review the information in WormBook on “Transformation and microinjection”. You might have a toxic sequence that you should inject at low concentrations, you might need to clean your PCR product differently (see the recent Worm Breeder’s Gazette), you might not have enough promoter. Have you tried rescuing with a cosmid, just to be sure that the mutant phenotype is really associated with your gene region?
Good luck, Janet