Name: NIA Mouse Embryonic Stem (ES) cell (Lif+, 48 h, high density) cDNA library (Long) Library ID: 2138 Organism: Mus musculus Strain: 129Sv/EvTac Gender: male Age: 0 Organ: embryonic stem cell Tissue: 129.3 ES cell line Host: DH10B Vector: pCMV-SPORT6 Vector type: phagemid Insert digest: 5' SalI/NotI 3' Stop Codon Status: without Description: This is a long-transcript enriched cDNA library (Ref. Genome Res. 11: 1553-1558 (2001). [PMID: 11544199]). ES cells were plated at density 3x104/cm2, on gelatin-coated plates and cultured for 48 hrs at 37 OC, 5% CO2. Culture medium: DMEM supplemented with 15% FBS, 2 mM L-glutamine, 0.1 mM NEAA, 1mM Sodium pyruvate, 0.1 mM beta-mercaptoethanol, 1000 U/ml LIF, 100 U/ml penicillin, and 100 ug/ml streptomycin. Double-stranded cDNAs were synthesized with an Oligo(dT) primer [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'] from 25g 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 pCMV-SPORT6 plasmid vector. The DH10B E. coli host was transformed with the ligation mixture by the standard chemical method. The average insert size is about 2.7 kb. The library was constructed by Yulan Piao.
