This cDNA library (plasmid DNA) is constructed from poly(A)+ RNA derived from Schizosaccharomyces pombe, strain h-L972 after treatment with HU(hydroxyurea), γ-radiation, and MMS(methylmethane sulfonate). It is constructed 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 pLZ3 vector used in this library can replicate both in S. pombe and in E. coli, and express cloned genes not only in S. pombe but also in mammalian cells as it contains SV40 promoter. It also contains f1 ori which is necessary for ssDNA synthesis, and bacteriophage T7 and T3 promoter for RNA synthesis (see Figure and Ref.2).
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. Tanaka,S. and Nojima,H. ”Nik1: a Nim1-like protein kinase of S. cerevisiae interacts with the Cdc28 complex and regulates cell cycle progression.” Genes Cells 1, 905-921 (1996) PMID: 9077450
3. Sambrook,J. and Russell,DW. Molecular Cloning Chapter 11 ”Preparation of cDNA libraries and gene identification.” CSHL Press (2001)
1. 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.)
2. Cloning the cDNA by functional complementation of the corresponding mutant strains.
Quantity : 500 ng (40 ng/ul, 13ul) in 10 mM Tris-HCl-1mM EDTA (pH 7.5)
1) Number of independent clones : 7.7 x 106
2) Average insert size : longer than 1 kb
Fig. Structure of pLZ3 and the restriction sites
Ars is the S. pombe region required for replication in S. pombe, and Ori is an origin required for replication in E. coli
cDNA Library, S.pombe ; After + HU, (GAMMA), MMS h-L972
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.