Functional cDNA libraries from Drosophila embryos.

摘要: We have modified current methods to create a very efficient technique for cloning cDNAs in defined orientation, into plasmid vectors bearing phage SP6 and T7 polymerase promoters. First strand synthesis is primed at the poly(A) tail with 26-mer synthetic oligonucleotide linker/primer, RNA hydrolyzed cDNA tailed 10 15 dG residues. The then annealed two prepared vector fragments specific ends of (one dC10–15 other 14-nucleotide cohesive end complementary linker/primer). After ligation second synthesized large fragment DNA I. Libraries up 8 × 106 independent transformants been obtained from 1 μg Drosophila poly(A)+ RNA. design method careful optimization first permitted several (4.3 6.5 kb), low abundance cDNAs. Transcription essentially full-length clones produces RNAs that are efficiently translated vitro give complete, unfused products, thus permitting rapid characterization via encoded polypeptides. Antisense can also be produced by transcription polymerase.