Catalog #: 10008
High Quality Normalized cDNA Libraries are produced using our primary cDNA libraries. Biotinylated driver cRNA produced from the T7 RNA polymerase promoter and single-stranded (ss) target cDNA produced from the F1 ori are hybridized to each other at a low Cot (concentration of driver times the time of hybridization) value. The cRNA:cDNA hybrids are removed by phenol extraction and the remaining ss target cDNA is converted to double-stranded DNA (dsDNA) with a repair oligo and Taq DNA polymerase. After electroporation of the dsDNA into T1 phage resistant E. coli, normalized clones are produced.
The normalization process reduces the level of abundant and moderately abundant genes and enriches for rare genes. To determine the reduction of an abundant gene, we compare the relative representation of the actin gene in the primary library with the normalized library by hybridization of different dilutions of the primary and normalized dsDNA with an actin probe. Using this method, we determine that the actin gene is reduced at least 20 fold in the normalized library.
We guarantee at least 1×106 normalized clones with > 87% recombinant clones containing an average insert size of at least 1 kb for standard normalized libraries and 3 kb for large insert normalized libraries. However, we generally produce libraries of 3×106 normalized clones with >95% recombinant clones containing an average insert size of 1.5 kb average insert size for standard normalized libraries and 4 kb for large insert normalized libraries.
Quality testing: Percentage of recombinant clones and average insert size determined by gel analysis of 24 clones picked at random. Actin gene reduction determined by hybridization of primary and normalized library.
Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at -80°C is recommended.