Has anyone had success using pools of fosmids from the Vancouver library for transgenic rescue of a mutant phenotype? Specifically, should almost every line made with a pool of ~6 fosmids rescue or is there variability? I remember when using pools of YACs or cosmids you sometimes had to try 10 or more lines to get rescue (assuming your gene is actually in the covered area). I have tried 3 lines for each fosmid group with no success. I want to make sure that this number is good enough to say that my gene isn’t in one of the areas covered by the fosmid pools. In my experience, I get rescue with every line when using one fosmid (that actually includes a particular gene) at a time but I’ve never used pools.
I am making standard extrachromosomal arrays using 10 ng / ul of each fosmid and 100 ng / ul of pPD118.20 (myo-3::GFP).
Thanks for your help!
Tim Kroft