title: A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein.
abstract: An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.
mesh_terms: D000818:Animals; D002460:Cell Line; D002466:Cell Nucleolus / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D011088:DNA Ligases / Q000235:genetics; D004722:Endoribonucleases; D005260:Female; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D008970:Molecular Weight; D016914:Ribonuclease H; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H; D001616:beta-Galactosidase; D011088:DNA Ligases