Description
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 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 NotI and BamHI (BglII)-SmaI 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 an SV40 promoter. It also contains an f1 ori which is necessary for ssDNA synthesis, and bacteriophage T7 and T3 promoters for RNA synthesis.
Application 1: PCR cloning of known or unknown genes.
Prepare primers for known or unknown genes (cDNAs) and amplify 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.)
Application 2: PCR cloning of cDNAs by functional complementation of corresponding mutant strains.
