Skip Main Navigation


Sequencing Info

Other Species Collections

Related Links

Useful Links

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.


  • 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.