"System 48" high-throughput cloning and protein expression analysis.
We describe a plate-based cloning and expression strategy for efficient high-throughput generation of validated expression clones in Escherichia coli. The process incorporates 48- or 96-well plates at all stages including the cloning and colony selection phases which are often performed manually. A 48-grid agar growth plate has been integrated into the colony selection component to improve throughput at the cloning stage. The combinations of 48- and 96-well plate formats are compatible with automated liquid handlers and multichannel pipettes. This revised cloning and expression pipeline increases throughput significantly, and also results in a reduction in both time and material requirements. The system has been validated by the production and screening of several thousand clones at the Midwest Center for Structural Genomics.
Abdullah
James M
JM
Biosciences Division, Argonne National Laboratory, Lemont, IL, USA.
Joachimiak
Andrzej
A
Collart
Frank R
FR
D000362:Agar / Q000737:chemistry; D000818:Animals; D018929:Cell Culture Techniques; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015203:Reproducibility of Results; D012995:Solubility; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D000362:Agar; D004247:DNA