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pRKW2 Library Method Overview

Dr. Wei Wu and Prof. Christof Niehrs constructed normalized libraries in the pRKW2 vector. Briefly, the method is as follows:
  • First strand cDNA was synthesized from poly A+ RNA using Invitrogen superscriptTM II RT and oligo dT primers
    (GA)5AGGATCC(T)16VN (where V=G, A, C and N=G, A, T, C), each containing a BamHI site. 5-methyl-dCTP was used instead of dCTP in this first-strand synthesis forming hemimethylated cDNA.
  • Full-length first strand cDNAs were enriched by capture of the biotinylated mRNA CAP structure by magnetic beads.
  • The mRNA was hydrolyzed, releasing first strand cDNA from the magnetic beads.
  • An oligo-dG tail was attached to the 3 end of the first strand cDNA using termianl deoxynucleotidyl trandferase (Invitrogen) and the strands were normalized against mRNA.
  • Second strand cDNA was synthesized using ExTaq poymerase (Takara) and primers (GA)5CTCGAGTTAATTAATC13, containing an XhoI site.
  • dsDNA was digested by XhoI/BamHI and cloned into the pRKW2 vector with XhoI at the 5 end and BamHI at the 3 end.

References:

  • Carninci P, et al., Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res. 2000, 10:1617-3.
  • Carninci P, Hayashizaki Y. High-efficiency full-length cDNA cloning. Methods Enzymol. 1999;303:19-44.