Protein Expression Plasmids Produced Rapidly: Streamlining Cloning Protocols and Robotic Handling

• DOI: 10.1089/adt.2005.3.661
• Publication Date: 2006-01-26T12:07:31Z
• Authors: Maria Kornienko; Allison Montalvo; Brian E. Carpenter; Michael Lenard; Pravien Abeywickrema; Dawn L. Hall; Paul L. Darke; Lawrence C. Kuo
• Keywords: Cloning (programming); Primer (cosmetics); genomic DNA

As many processes in the preclinical drug discovery process become highly parallel, need to also produce a large number of different proteins parallel has acute, such as for protein crystallization and activity screening. In turn, requisite DNA constructions these must now be done at rate that requires automated cloning procedures, each with an intrinsic low failure probability per sample. The high-throughput solutions presented here achieve production 192 expression plasmids success greater than 95% targeted open reading frames. Time completion set by one person is reduced approximately 11 working days, starting polymerase chain reactions source clones ending purified plasmids. Achievement this throughput utilizes following: (1) Beckman Coulter (Fullerton, CA) Biomek FX liquid handler most manipulations, (2) Gateway technology (Invitrogen Corp., Carlsbad, CA), (3) computer programs designed processing all sample information, including primer design resulting sequence assembly. Exemplary data are form Rho-kinase crystallizes (ROCK2).