A serial dilution is a chain, and a chain carries its errors forward. Each step takes a portion of the last, so a small mistake early on, a little carryover or an incomplete mix, is amplified by every step that follows. Getting a dilution series right is less about any single transfer and more about not letting small problems compound.
Fresh tips every step
Reusing a tip across dilution steps drags concentrated sample into more dilute wells, which is exactly the carryover a dilution series cannot tolerate. Use a new tip for every transfer. It is the simplest defense against a contaminated curve.
Mix well, but not too full
Each step needs the added sample fully blended into the diluent before you draw from it, or the portion you carry forward is not actually at the target concentration. Mix thoroughly, starting with several mix cycles and trimming only if speed demands it. Keep the mix volume below about 80 percent of the well volume, though, because mixing too close to full pulls in air and makes bubbles.
Drop the over-aspirate
Over-aspiration helps a single accurate transfer, but in a dilution it works against you: the extra volume drawn and returned is a route for carryover into the next mixing step. Remove the over-aspirate volume for dilution classes so you are not quietly shuttling excess between wells.
Mind level detection and following
If you rely on liquid level detection and the tip following the surface, the labware definition has to be accurate, or the tip mis-tracks during the many small transfers a dilution involves. Mixing at a fixed, sensible height also keeps the dispense and mix consistent from step to step.
- Use a new tip for every transfer.
- Mix thoroughly, keeping mix volume under about 80 percent of the well.
- Remove the over-aspirate volume to avoid carryover.
- Dispense to the surface where possible, and verify labware if using level following.
In a dilution, every step inherits the mistakes of the last. Spend your care on clean tips and complete mixing, and the curve takes care of itself.