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PubMed · 1849863

High-yield method for directional cDNA library construction.

G Bellemare·C Potvin·D Bergeron
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Abstract

Improvement of a cDNA synthesis procedure using a single stranded (ss) vector primer [Bellemare et al., Gene 52 (1987) 11-19] is reported. This vector (pPBS27), upon linearization with XbaI using an appropriate restriction site-directed fragment, releases a thymidilic tail used to prime cDNA synthesis. DNA polymerase I and RNase H replace the RNA strand and replicate the vector before double-stranded (ds) blunt-end ligation with T4 DNA ligase. More than 10(7) cfu/microgram of vector can be obtained with an efficient transformation protocol using either globin-encoding or 7.5-kb poly(A)-tailed RNA. This improved cloning method is easier, faster and a few hundred times more efficient than the original procedure as it involves ds rather than ss DNA for transformation.

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Base SequenceCloning, MolecularDNADNA Polymerase IDNA, RecombinantDNA, Single-StrandedEndoribonucleasesGene LibraryGenetic VectorsGlobinsMolecular Sequence DataOligonucleotide ProbesPlasmidsRestriction MappingRibonuclease HJournal ArticleResearch Support, Non-U.S. Gov't

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G Bellemare, C Potvin, D Bergeron. 1991-02-15. High-yield method for directional cDNA library construction.. https://doi.org/10.1016/0378-1119(91)90178-e

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