Author: Rogers, J.; Schoepp, R.J.; Schröder, O.; Clements, T.L.; Holland, T.F.; Li, J.Q.; Li, J.; Lewis, L.M.; Dirmeier, R.P.; Frey, G.J.; Tan, X.; Wong, K.; Woodnutt, G.; Keller, M.; Reed, D.S.; Kimmel, B.E.; Tozer, E.C.
Title: Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases Document date: 2008_5_13
ID: xkx56h0o_15
Snippet: For confirmation of clones isolated from the discovery screen, recovered plasmids were retransformed into the Rosettagami strain and plated on agar. Approximately 100 -500 single colonies were selected and placed into 96-well plates containing 0.2 ml LB-Carb. The cells were grown for 16 h, and then diluted 50-100-fold into 1 ml LB-Carb. The diluted cells were grown and induced at middle log phase with 0.2% arabinose and 100 mM ZnCl 2 for 3 -12 h .....
Document: For confirmation of clones isolated from the discovery screen, recovered plasmids were retransformed into the Rosettagami strain and plated on agar. Approximately 100 -500 single colonies were selected and placed into 96-well plates containing 0.2 ml LB-Carb. The cells were grown for 16 h, and then diluted 50-100-fold into 1 ml LB-Carb. The diluted cells were grown and induced at middle log phase with 0.2% arabinose and 100 mM ZnCl 2 for 3 -12 h at 208C. Cells in 96-well plates were pelleted by centrifugation for 20 min at 4000 r.p.m., resuspended in 125 ml sonication buffer, and transferred to a skirted 96-well PCR plate (Fisher Scientific, Pittsburgh, PA, USA). The cells were sonicated for 1 min in an ice-bath chilled microplate horn (Misonix, Farmingdale, NY, USA). The sonicated plates were centrifuged at 1000 r.p.m. for 25 min and the lysate added directly onto an antigen-coated 96-well ELISA plate.
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