Ni-NTA Superflow 96 BioRobot Kit (4)

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Fragment library screening: For screening, plasmids with inserts were used to transform E. coli BLR(DE3) cells, and picked colonies were grown in 24-well blocks containing LB media supplemented with Overnight Express Autoinduction System 1 (Novagen) at 37°C for 12 h. Aliquots from each well were aggregated into 96-well blocks and lysed with 2 μg/mL RNaseA (AbGene), 0.6 μg/mL DNase I (Roche), and 2.5 μg/mL lysozyme (Sigma) at 30°C for 1 h.
To determine “in-frame” expression, lysates were “dotted” onto a Protran nitrocellulose membrane (Schleicher & Schuell), which was then probed with Anti-His6 mAb (BD Biosciences) and developed with anti-mouse IgG-AP Conjugate (Promega) and BCIP/NBT (Sigma). Dark spots on the membrane were registered as positive expression hits.
A second aliquot of each culture was transferred to a new 96-well block, which was centrifuged, and the pellets were stored at −20°C for later DNA analysis. The remaining cultures were spun down, and the supernatants were discarded.
To determine soluble expression, lysates were subjected to filtration and affinity purification using the Ni-NTA Superflow 96 BioRobot Kit (QIAGEN) on a BioRobot 8000 (QIAGEN), and the eluate was dotted onto nitrocellulose membrane for immunodetection, as above. Dark spots on the membrane were registered as potential soluble hits. Aliquots of these samples were run on each of two SDS-PAGE gels—one stained with Coomassie Brilliant Blue R (Sigma) and the other blotted onto nitrocellulose membrane for immunodetection as described above. Clones presenting visible and correlated bands in both detection modes were registered as positive soluble hits. The gene fragmentation and screening process that comprises CDH is the subject of the published patent application WO 03/040391.



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