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ESTs provided by our cooperation partners are transferred as bacterial colonies either in glycerol stock or on solid medium, purified in our labs under strict QC measures, and stored as plasmid DNA as well as PCR products at -20°C in the PICME database. Sequencing of the plasmids is performed in consecutive steps.
Production process QC
After delivery, material is stored at 4°C until data have been verified and processed into the PICME DB. Bacteria are grown in liquid media. Plasmid DNA is prepared in 96 well format. The correct orientation of the plates is checked by sequencing of 3 samples per plate. All plates are barcoded when entering the production process.
Purified plasmid DNA is transferred to 384 master plates and onto Clonesaver Cards (as a backup), and dilutions for PCR are prepared; 100µl PCR reaction is prepared from the plasmid DNA. PCR products are analysed on agarose gels and documented using the in-house software GelMasterTM. PCR products are aliquoted in replica plates.
At this point in time material is already available for spotting, but currently has no sequence verification. Subsequently ESTs are selected according to their length and preliminary annotation for de novo sequencing. These sequence-verified clones can be used for unigene clustering analysis.
PCR products for chip production are not purified, but they have to match quality measures in amount (at least 200ng/µl) and purity. PCR products are spotted in SSC buffer, supplemented with Betaine on GAP slides from Corning. Negative/positive controls have to be added specifically upon demand. Spotted arrays are checked by scanning, batches are checked by oligo hybridisation for spot morphology and signal to noise ratio. All QC steps are documented.