A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein.
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.
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
D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H; D001616:beta-Galactosidase; D011088:DNA Ligases