cbot cluster generation

[dsidote]

HI,

Since receiving our cbot we have observed this pattern of uneven cluster density. We are using 8pM input. Has anyone else observed this pattern and/or know what's causing this?

Dave

[jfostel]

Higher density at the inlet side of the flowcell is pretty common and can happen on either Cluster Station or cBot. Everything involved in the cluster en reaction (sample, polymerase, nucleotides, etc) will be at a slightly higher concentration at the inlet before a lot of binding and mixing inside the flowcell occurs.

[lindsayu]

I have been investigating this phenomenon in hopes of "evening-out" the cluster density down the flowcell. Our hope is that by attaining a more uniform cluster density perhaps our passing filter rates could be improved, we could have more usable data, and just be more efficient overall.
We think this phenomenon results from there being a greater concentration of free (unbound) template at the front end of the flowcell than there is at the end. Somehow we need to maintain an even distribution of free template across all parts of the flowcell. This is obviously impossible since template will bind to the oligos on the flow cell's surface as soon as they come into contact, but how can we optimize this? Our current hypothesis is that the cBot template (DNA) pump speed could be altered to minimize the uneven template concentration. Has anyone attempted this?