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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.
- 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.