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The fact that the target isoform PI3Kα was a poor template for screening these compounds compared to PI3Kγ and PI3K δ was somewhat surprising.
The concentrations of the DNAs were measured with a Nanodrop 2000 spectrophotometer and they were diluted to have a concentration of 5 ng/ μL with deionized-distilled water and used as a PCR template for screening.
Because there is no obvious a priori approach to choosing a single structural template for screening a compound that may bind to multiple distinct motifs, our results suggest that using as wide an array of diverse templates as appropriate should be considered when running SMAP screens.
The general template for screening of formulation for insoluble NCEs at discovery stage has been described in the literature.
Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potential gun4 complements.
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DNA samples were then pooled (four samples per well of a 384-well plate) and the DNA pool was used as a template DNA for screening.
The consortium developed protocols, templates, and checklists for screening abstracts/papers retrieved in the literature searches to ensure a homogenous process across the research questions and across reviewers.
Since it is desirable in terms of throughput to screen large amplicons that contain mostly coding sequences, DNA templates are best suited for screening large single exons, or two to three small exons separated by small introns.
The latter tubes were centrifuged to remove the LB broth and cells were resuspended in 15 μl of pH 7.0 Tris-HCl buffer. 1 μl of resuspended cells was added as template to PCR reaction tubes for screening.
Although the yield of template is low, this step allows for screening large numbers of proteins.
Adjustments in the injection voltage and injection time were occasionally necessary for screening the cDNA templates due to the variability in the amount of RT-PCR products in multiplexed reactions, which is inherent in the differences in the expression levels for different genes.
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