High Quality Subtracted cDNA Libraries are produced using our primary target and driver 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 high 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, subtracted clones are produced.
The subtraction process removes genes that are in common (i.e., actin) between two different libraries (i.e., normal versus disease) and enriches for unique and highly induced genes. To determine the effectiveness of the subtraction process, we compare the relative representation of the actin gene in the primary library (target) with the subtracted library by hybridization of different dilutions of the primary and subtracted dsDNA with an actin probe. Using this method, we determine that the actin gene must be reduced at least 20 fold in the subtracted library. Generally, we see at least 100 fold reduction of the actin gene in the subtracted library.
We guarantee subtracted libraries of at least 1×105 subtracted clones with > 87% recombinant clones containing an average insert size of at least 1 kb. However, we generally produce libraries of >5×105 subtracted clones with >95% recombinant clones containing an average insert size of 1.5 kb.
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 subtracted libraries.
Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at –80°C is recommended.