one bead-one fragment?

[jean_valjean]

Anybody knows how this one beadne fragment ratio is guaranteed before emPCR? In other words, how is the ligation of multiple fragments to just one bead avoided. Roche says that titration ensures the ratio but I did not understand. In almost every publiction, it says "in conditions favoring the binding of only one fragment to individual beads" but no details are given. A book says that DNA concentration is kept very low with respect to bead amount. Which one is correct?

[flxlex]

Titration is done to find a bead-DNA ration that minimizes the two-DNA-molecules-per-bead amount. So, it is pure dynamics and stochastics. There might be something special in the buffer but I can't imagine what that would be. That's also why after emPCR, bead recovery and enrichment, one gets anywhere from a few to maybe 20% recovery of beads with DNA on them. Such a waste...

[jean_valjean]

one bead - one fragment

Thank you very much for your help

[epistatic]

What you are shooting for is a double poisson distribution. To have the one-bead and one library DNA molecule in a droplet you need the majority of droplets be empty.