A simple and very efficient method for generating cDNA libraries
作者： Ueli Gubler , Beth J. Hoffman
DOI: 10.1016/0378-1119(83)90230-5
关键词:
摘要: A simple method for generating cDNA libraries from submicrogram quantities of mRNA is described. It combines classical first-strand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161-170]. Neither elaborate vector-primer system nor hairpin loop cleavage by S1 nuclease are used. thus made can be tailed cloned without further purification or sizing. Cloning efficiencies as high 10(6) recombinants generated per microgram mRNA, a considerable improvement over earlier methods. Using fully sequenced 1300 nucleotide-long bovine preproenkephalin we have established sequencing that yields faithful full-length transcripts. This procedure considerably simplifies establishment cloning low-abundance mRNAs.
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