![]() After a two-round screening, the randomized sequences were substantially converged to peptide-encoding sequences recognized by the anti-His6 antibody. The products of emulsion PCR are loaded onto a special pico titre plate. Step 1: genomic DNA is sheared and then blunt-ended. Each water droplet initially contains only a magnetic microbead wrapped in a. A library with a theoretical diversity of 10(6) was constructed by randomizing the middle four residues of the His6 tag. Overview of genomic library construction on microbeads using emulsion PCR. After incubation with fluorescein isothiocyanate (FITC)-labeled anti-His6 (C-term) antibody, the beads with the His6 gene were enriched 917-fold in a single-round screening by using flow cytometry. We mixed two types of DNA templates of Histidine6 tag (His6)-fused and FLAG tag-fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. The pool of beads was then subjected to cell-free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti-glutathione S-transferase (GST) antibody. The template molecules were amplified and immobilized on beads via bead-linked reverse primers and biotinylated forward primers. A PCR mixture containing streptavidin-coated microbeads was compartmentalized by water-in-oil (w/o) emulsion with estimated 0.5 template molecules per droplet. Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. Abstract: BEAMing (beads, emulsion, amplification, and magnetics). We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. An emulsion digital PCR quantitative method based on microbeads and micropillar array chip. ![]()
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