cDNA library S.pombe strain h-L972 after treatment with HU(hydroxyurea) gamma-radiation and MMS

This cDNA library (plasmid DNA) is constructed from poly(A)+ RNA derived from Schizosaccharomyces pombe strain h-L972 after treatment with HU(hydroxyurea) -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). Application: 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.). Cloning the cDNA by functional complementation of the corresponding mutant strains.