This cDNA library (plasmid DNA) is constructed from human embryonic kidney 293T cell -derived poly(A)+ RNA by the Linker-Primer method (Ref.1) by Professor Hiroshi Nojima of Research Institute for Microbial Diseases, Osaka University. This library is unidirectionally cloned by using the oligo (dT)18 linker primer which contains the restriction enzyme site of Not I, and BamHI (Bgl II)-Sma I adaptor.
The pAP3neo vector used in this library can express rat genes in mammalian cells as it contains SV40 promoter. It also contains Ori of pUC plasmid required for replication in E.coli, f1 ori which is necessary for ssDNA synthesis, and bacteriophage T7 and T3 promoters for RNA synthesis (see Figure). GenBank Accession No. AB003468
1. Kobori,M., Ikeda,Y., Nara,H., Kato,M., Kumegawa,M., Nojima,H., and Kawashima,H. ” Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.” Genes Cells 3: 459-475 (1998) PMID: 9753427
2. Sambrook,J. and Russell,DW. Molecular Cloning Chapter 11 ”Preparation of cDNA libraries and gene identification.” CSHL Press (2001)
PCR screening of known or unknown gene: Prepare the primers for the known or unknown gene (cDNA) and amplify the gene by PCR from this library followed by cloning to an appropriate vector. It is useful for large-scale protein productions, and preparation of probes, etc.
Standard amplifying conditions: 35 cycles of PCR reactions using 10-100 ng of cDNA as a template. (Change the quantity of template and the number of cycles depending on the expression rate of mRNA of the objective gene.)
Quantity : 500 ng (40 ng/ul, 13ul) in 10 mM Tris-HCl-1mM EDTA (pH 7.5)
Quality : 1) Number of independent clones: 2.2 x 106
2) Average insert size : longer than 1 kb
Fig. Structure of pAP3neo and the restriction sites
cDNA Library, Human ; 293T