124. Generation of Large-Insert Mouse cDNA Libraries 

Lisa Stubbs¹, Jimmy Spearow², and Xiaojia Ren^1 
1DOE Joint Genome Institute and Human Genome Center, Lawrence Livermore National Laboratory, Livermore, CA 94550 and ²Section on Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616 
stubbs5@llnl.gov 

We have developed straightforward, reliable and efficient protocols for generating large-insert clone libraries from size-selected cDNA. We have found enzyme combinations and RNA preparation protocols that routinely produce double-stranded cDNA products with excellent representation of very large cDNA fragments (5-15 kb). After cDNA synthesis, the products are fractionated on sucrose gradients, and each size fraction is cloned and plated as a separate sub-library. Size fractions are chosen for screening according to information obtained from Northern blot analysis or from PCR screening of pooled sublibrary clones. Our most recent efforts, funded as part of the JGI functional genomics pilot program, have focused upon improvements in methods of library production and screening; we have also begun to experiment with new methods to normalize large insert pools before cloning. Using these improved protocols, we have recently created a series of new mouse cDNA libraries representing brain, thymus, ovary, and other mouse tissues. We will work with I.M.A.G.E. to share these resource libraries with the Genome research community as widely as possible, to serve as a resource for isolation of full-length cDNA clones.