BioSample

Mouse cDNA project by the Laboratory of Genetics, National Institute on Aging (NIA), Intramural Research Program, NIH (http://lgsun.grc.nia.nih.gov/cDNA). This is a long-transcript enriched cDNA library (Ref. Genome Res. 11: 1553-1558 (2001). [PMID: 11544199]). Total RNAs were extracted from a pool of 13 embryos at 8.5-days postcoitum. Double-stranded cDNAs were synthesized with an Oligo(dT) primer [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'] from 9.1 ug of total RNA, treated with T4 DNA polymerase, and purified by ethanol-precipitation. The cDNAs were ligated to Lone-linker LL-Sal4, purified by phenol/chloroform, and separated from free linkers by Centricon 100. Then, the cDNAs were amplified by long-range high fidelity PCR using Ex Taq polymerase (Takara) with a primer Sal4-S. The products were purified by phenol/chloroform and Centricon 100. The cDNAs were digested with SalI and NotI enzymes and cloned into SalI/NotI site of pSPORT1 plasmid vector. The DH10B E. coli host was transformed with the ligation mixture by the standard chemical method. The average insert size is about 2.5 kb. The library was constructed by Yulan Piao (NIA).