Construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent vector.

摘要: We have designed a simple procedure for the construction of directional cDNA libraries enriched full-length inserts in transcription-competent cloning vector. An oligonucleotide, its 5' end starting with heteropolymeric sequence encoding rare restriction sites NotI and SfiI, followed by 50 dT residues, is used to prime first-strand synthesis on size-selected mRNA. After second-strand EcoRI linker addition, double digested NotI, or generate DNA fragments asymmetric ends that can be directionally cloned. The are "full length" size selection ligated into phage lambda vector containing T3 T7 RNA polymerase promoters. These directly vitro sense antisense RNA.