Library replication using the Genetix QBot
(a) Before replication is started, microtiter plate holding blocks are mounted on the main bed of the QBot, and the UV lamps are used to sterilize the working area in the QBot. After UV-sterilization, the wash bath is filled with 70-100% ethanol, and twenty-four 384-well plates containing sterile freezing media are placed on the microtiter plate holding blocks. The plate lids are removed. Hotels containing the first 72 plates of the master copy of the library (36 plates per hotel) are fitted onto the hotel holders. The "Replicating" software application is opened. At this point, the instrument is ready to begin replication. See APPENDIX D for a diagram of the QBot and APPENDIX E for specific details on general operations of the QBot (e.g., pin sterilization, plate manipulation, and inoculation).
(b) At the start of the replication cycle, the replicating head (made up of 384 pins) is sterilized and dried by the ethanol bath and air-drier, respectively. The robotic arm transfers master copy (MC) plate 1 from the first plate slot in the right hotel to the plate holder. The lid is removed from MC plate 1 by the lid manipulator (see APPENDIX E for details on these processes). The replicator head is then positioned over MC plate 1 so that each pin is directly over a corresponding well. The arm is lowered simultaneously inoculating all 384 pins of the replicator head. The robot arm is raised, and quickly moved into position directly over the plate in position 1 of microtiter plate holding BLOCK A. A quick downward movement of the robot arm inoculates the plate (which should be labeled Copy 1, plate 1). The picking head is then re-sterilized as described above. The lid is replaced on MC plate 1, and the plate is returned to the hotel slot from which it was removed. This marks the completion of the first replication cycle. The second replication cycle results in replication of MC plate 2, the third in MC plate 3, etc. After 24 complete cycles, all plates on the main bed have been inoculated. At this point, the QBot pauses, and the user can place 24 new microtiter plates on the main bed. Clicking on the "continue" icon on the computer screen will resume the QBot's activities (starting with the replication of MC plate 25).
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