Construction of cDNA libraries by blunt-end ligation: High-frequency cloning of long cDNAs from filamentous fungi
✍ Scribed by Tuula T. Teeri; Vijay Kumar; Päivi Lehtovaara; Jonathan Knowles
- Publisher
- Elsevier Science
- Year
- 1987
- Tongue
- English
- Weight
- 1022 KB
- Volume
- 164
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to lo6 clones/pg mRNA, is described. Routine synthesis oftranscripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma rt?eSf?i CehhX.S.