Emulsion PCR and Bead Enrichment
Emulsion PCR (ePCR)
In emulsion PCR (ePCR) clonal bead populations are generated in water-in-oil microreactors containing 1.6 bln P1-coupled beads, template, PCR components, and primers. Templates (either a fragment or mate-paired library) are amplified approximately 30,000 times in these 10-µm microreactors. In this process, a polymerase extends from the P1 adapter after the template anneals to the P1-coupled beads. A complementary sequence is extended off bead surface followed by template dissociation.
Enrichment of Templated Beads
After ePCR, the templates are denatured and a bead enrichment step is performed in a glycerol gradient to separate beads with extended templates from the unwanted non-templated beads. P2-coated Polystyrene beads are used to collect P2-Positive templated beads, recovering approximately 200 Million beads, or 80% of the total P2-Positive beads.