How a run comes together
Three stages. The builder keeps your work in the browser between sessions, and Save writes a file you can reopen later.
Pick the instrument, set the deck, add your steps. Problems show up as you go — red must be fixed, amber is worth a look.
The .export52 for epBlue, and a setup sheet you can print or open on a tablet at the bench.
Import into epBlue, set the deck up as the sheet shows, choose automatically detect tools, and enter the sample count from the sheet.
The six tabs
Work left to right. A pooling run only needs Deck, Pool and Review.
- RunThe instrument, how many samples (1–8, one column), and which plate column they are in. Mark any columns that are already used and you will be warned if a step would put liquid into one.
- DeckDrag labware onto the positions, or tap an item and then tap a position. Positions use the codes engraved on the deck — never software slot numbers.
- ReagentsName each reagent and say what kind of liquid it is; that picks the pipetting profile. Put it in a tube or a trough and the load volume is worked out for you.
- StepsAdd steps from the menu. Bead clean-up expands into beads, binding, magnet, washes, drying and elution. Plate moves, magnet waits and tip changes are added for you.
- PoolBuild a library pool from concentrations alone. See below.
- Review & exportThe live checks, a step-by-step walkthrough whose row numbers match epBlue's command list, and the two downloads.
Pooling libraries
Say where the samples are, type in what you measured, and say what you are aiming for. The builder works out how much of each library to take, adds the buffer, and writes the transfers into the protocol — no worklist CSVs to load at the instrument.
Samples in any wells of the plate, or in 1.5 mL tubes; the pool goes into one tube or one free well. A pool can draw from up to 96 wells or 24 tubes.
A pool concentration and volume, a total in fmol, a set amount from each sample, or simply the same volume from each. Illumina and ONT presets fill in starting points.
A relative share per sample: 1 everywhere is equimolar, 2 gives that library twice the share, 0 leaves it out. You see the share each sample really ends up with.
ng/µL or pg/µL with an average fragment size, or straight as nM. The maths matches PoolMate, so the two programs never disagree.
One dilution step can only divide by so much, so anything stronger is taken down in stages — each into its own well or tube, and mixed before the next one draws from it. A library at 1400 nM headed for a 2 nM pool becomes two steps:
| Step | Take | Buffer | Into | Factor | Ends at |
|---|---|---|---|---|---|
| 1 of 2 | 2 µL from A1 | + 18 µL | A10 | 1:10 | 140 nM |
| 2 of 2 | 2 µL from A10 | + 18 µL | B10 | 1:10 | 14 nM |
The free wells and tube positions on your deck are counted first. If the dilutions need more room than the run can spare, the builder says so — and can write them as a separate run first, which also lets you re-quantify before you pool. You then get two protocols, each with its own setup sheet, and the pooling sheet tells you which to run first.
Checks before you can export
The builder will not export a protocol with a problem in it. Among other things, it checks:
- that the plate is sitting on something that can hold it, and that labware and reagents are where the steps expect them
- that every volume is inside the range of a tool that is actually on the deck, and that ethanol only ever uses the 300 µL tool
- that nothing shakes while hot, and that a cold hold is released rather than left cold
- that no step puts liquid into a column you marked as already used
- that the last wash comes off at the bottom of the well, never below it
Planning at the desk with PoolMate
PoolMate plans pools, normalisations and dilutions at the bench. Press Generate worklists there, keep the ..._pool.poolplan.json, then open it here with Pool → Import plan / CSV — and a pool planned at the desk becomes a run. The builder saves the same file back, so a plan travels either way.
Before you trust a run
Not yet run on the instruments
- Pooling, including the dilutions it does inside the run
- The 3-trough reservoir rack on the 5075 — the builder flags it
- Tempering C2 below 10 °C on the 5073t — the builder warns
Watch the first of these runs and use material you can afford to lose. More than 8 samples in a column workflow is not supported; pooling is not limited that way.
At the instrument
- In epBlue, import the
.export52. - Set the deck up exactly as the setup sheet shows. Take the caps off the tubes and empty the liquid waste.
- At run setup, select automatically detect tools. If you skip this, epBlue reports that a tool position is occupied.
- When epBlue asks for the number of samples, enter the number on the sheet.
- In the Volume dialog, nothing should be outlined in red.
If epBlue reports an error at a command number, look at that row in the walkthrough on the Review tab — the row numbers match.