Canadian Forest Service Publications

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors. 1988. Rutledge, R.G.; Seligy, V.L.; Côté, M.J.; Dimock, K.; Lewin, L.L.; Tenniswood, M.P. Gene 68(1): 151-158.

Year: 1988

Issued by: Laurentian Forestry Centre

Catalog ID: 26013

Language: English

Availability: Not available through the CFS (click for more information).

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We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263–269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.