Name: DFCI-Kazusa.orf Library ID: 2463 Organism: Homo sapiens Age: 0 Organ: unspecified Host: DH5alpha T1-resistant Vector: pDONR223 Vector type: plasmid Insert digest: 5' attL1.1/attL2.1 3' Stop Codon Status: without Description: Clones from the Kazusa DNA Research Institute were generously donated for use as the starting template. 8 ul of each clone was inoculated in 1 ml LB containing the appropriate antibiotic. A BP recombinational reaction contains 2 ul of 5 X BP3 buffer; 2 ul of BP clonase; 1 ul of pDONR223 (150 ng/uL); 2 ul of PCR product (2-200 ng/uL); 3 uL H2O. The 5 X BP3 buffer consists of 100 mM Tris-Cl (pH 7.5); 20 mM EDTA; 30 mM spermidine-HCL; 25 percent glycerol; 225 mM NaCl. LR reactions we performed as described previously with minor changes (Reboul et al. 2003, Rual et al. 2004). BP products were transformed into liquid cultures of E. coli, with antibiotic selection of spectinomycin at 50 ug/mL. Clone structure of the ORF and flanking sequences is as follows: 5'-gtacaaaaaagttgGCACC-ORF-TTGccaactttcttgtac-3' where lower case corresponds to the att sites and upper case corresponds to linker sequence. Clones from this library do not contain a stop codon. Library constructed by the Dana Farber Cancer Institute, Center for Cancer Systems Biology for the ORFeome Collaboration.