Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background.
A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.
D002482:Cellulose / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D006824:Hybridization, Genetic; D009838:Oligodeoxyribonucleotides / Q000302:isolation & purification; D010957:Plasmids; D014169:Transformation, Bacterial
D009838:Oligodeoxyribonucleotides; C033539:oligodeoxycytidylic acid; C033538:oligodeoxyguanylic acid; D002482:Cellulose; D004247:DNA