High-efficiency cloning of full-length cDNA.

Abstract

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 105 plasmid-cDNA recombinants obtained per ,ug of rabbit reticulocyte mRNA, about 10%o contained a complete a-or P-globin mRNA sequence, and at least 30 to 50%o, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full-or nearly fulllength cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first-and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor.

“…High-density screening of the oligo(dT)-primed peripheral blood lymphocyte library was carried out by plating about 10,000 colonies per 150-mm plate on a GeneScreen membrane (22). About 20 plates were screened with the oligonucleotide probe (see Results) for the signal peptidase cleavage site (see Fig.…”

“…The eukaryotic expression vector V19.10, predigested with EcoRI and HindIII, was ligated with the cDNA; ligation was followed by the transfection of competent Eschenchia coli DH5a cells (GIBCO-BRL). The cDNA library was frozen in aliquots at -80°C after the addition of dimethyl sulfoxide to 7% (22).…”

Restriction enzyme analysis of faecal adenoviruses in Newcastle upon Tyne

“…The remaining 35 gl of each sample was reprecipitated from 2 M ammonium acetate with ethanol (Okayama & Berg, 1982) and the pellet was resuspended in 80 % ethanol and repelleted. DNA was resuspended in an appropriate volume of distilled water, between 10 and 50 ,u depending on the intensity of the adenovirus DNA band on the gel.…”

Cloning of cDNA to rat mammary-gland fatty acid synthase mRNA. Evidence for the expression of two mRNA species during lactation

“…After purification by three passages through oligo (dT)-cellulose, poly(A) mRNA was size-fractionated on 5-20 % sucrose density gradients and the fraction of mRNA eluting with a molecular mass greater than 28 S ribosomal RNA was retained and used for construction of the cDNA library. Production of 'filled-in' double-stranded cDNA was performed using the protocol of Okayama & Berg [19] as modified by Gubler & Hoffman [20]. A random primed cDNA library was constructed using a Transcriptaid cDNA synthesis kit (P and S Biochemicals).…”