# Pre-Wetting Pipette Tips: Why It Matters and How to Do It Consistently Pre-wetting a pipette tip means aspirating and dispensing liquid into the tip once or twice before you deliver the volume you actually intend to measure. The maneuver takes two to five seconds per tip, adds essentially nothing to total elapsed time, and addresses one of the largest systematic errors in manual liquid handling: the volume difference between the first delivery from a dry tip and every delivery that follows. When you pre-wet consistently, your standards, samples, and replicates all experience the same tip condition, which removes a hidden bias from your data rather than merely reducing scatter. Use pre-wetting whenever accuracy matters: preparing standard curves, dilution series, qPCR master mixes, cell culture seeding densities, enzyme assays, and any workflow where a first-draw error would propagate into every downstream calculation. Skip it only in rough transfers where volume is approximate by design, for example resuspending a pellet or topping off a buffer to a beaker mark. Hands-on time is short. For a single pipetting event, pre-wetting adds about five seconds. For a full 96-well plate set up by hand, budget an extra five to ten minutes across the session. Total elapsed time is unchanged because the pre-wet steps happen inside pauses you already have, such as waiting for a centrifuge or an incubator. ## Why a Dry Tip Delivers the Wrong Volume ### The physics of the first draw An air-displacement pipette works by moving a piston a fixed distance, which displaces a fixed volume of air. The liquid that enters the tip is pushed in by that displaced air, so anything that changes the behavior of the air column or the liquid surface changes the delivered volume. A brand new, dry tip interferes in three ways. First, evaporation. The instant your sample enters a dry tip, part of it begins to evaporate into the dry air inside the tip barrel. Water leaves the liquid phase and becomes vapor, so the volume of liquid that leaves the tip is smaller than the volume the piston requested. This effect is largest for the first aspirate because the air inside the tip is at its driest. After one draw, the dead air space above the liquid is saturated with vapor and the effect largely disappears. This is the single biggest reason the first delivered volume from a dry tip runs low, and it is the core rationale for pre-wetting pipette tips. Second, surface retention. Liquid wets the inner wall of the tip and a thin film clings to the plastic after dispensing. On the first draw, part of the aspirated volume stays behind as this film, so the delivered volume runs low again. Once a film is established, each subsequent draw loses a similar, small amount to the film while also picking up a similar amount from the previous draw, so the losses cancel and delivery stabilizes. Pre-wetting establishes that film in advance, during a throwaway draw. Third, temperature. Cold liquid entering a warm tip, or warm liquid entering a cool tip, changes the density of the aspirate and can cause the meniscus to creep after the pipette has been removed from the liquid. Equilibrating liquids and tips to the same temperature, then pre-wetting, brings the tip into thermal contact with the liquid before the measured draw. ### Why consistency matters more than the first draw itself There is an important subtlety. If every volume in an experiment were pipetted from a dry tip, the systematic first-draw loss would partially cancel out, because standards and samples would all be biased in the same direction. The real danger is inconsistency. If you pre-wet the tips for your standards because you read this article, then rush the samples at the end of the day without pre-wetting, the two groups carry different systematic errors. A calibration curve built with pre-wetted tips applied to samples pipetted dry produces concentrations that are quietly wrong in a defined direction. The fix is not only to pre-wet, but to pre-wet the same way, every time, for every condition in the experiment. The same principle governs any multi-step measurement workflow. Studies of impression techniques in restorative dentistry show that the dimensional accuracy of the final cast depends on disciplined execution of every handling step, and that the accuracy of the most critical step dominates the outcome [1]. Work with optical three-dimensional scanning quantifies this idea: the systematic error, or bias, of a carefully controlled acquisition method sat between 14 and 21 micrometers, small in absolute terms but meaningful relative to the tolerances the procedure had to meet [2]. Pipetting follows the same logic. A first-draw error of a few percent in every standard is invisible to the eye but shifts every calculated concentration in your assay. ### How big is the effect? The magnitude depends on the volume, the liquid, and the environment. The relative impact of first-draw evaporation and retention is largest at small volumes, where a fixed absolute loss is a larger fraction of the target. A 2 microliter draw from a 2 to 20 microliter pipette loses proportionally far more to a dry tip than a 200 microliter draw from a 200 microliter pipette. Volatile solvents such as ethanol show a larger evaporation effect than aqueous buffers. Viscous liquids show a larger retention effect. Humid laboratory air shrinks the evaporation penalty, and dry winter air in a heated building enlarges it. For context on what good technique can achieve, validation work on automated pipetting systems for cell culture toxicology reported technical imprecision of about 2 percent for a cell survival assay and roughly 4.5 to 6 percent for bioluminescent and ELISA readouts, after the systems were validated for pipetting accuracy before use [3]. That level of precision is the benchmark that careful manual pipetting technique, including consistent pre-wetting, is trying to approach. Conversely, work that systematically optimized multi-channel pipetting in a kinase assay demonstrated that measurable gains in inhibition data quality come directly from tightening liquid handling practice rather than from changing the assay chemistry [4]. ## Materials and Reagents | Item | Specification | Working concentration or condition | Purpose | |--|--|--|--| | Air-displacement pipette | Single or multi-channel, calibrated within the last 6 to 12 months | Set to 25 to 100 percent of the nominal volume range | Volume delivery | | Pipette tips | Matched to pipette brand or verified compatible, sterile if working with cultures | Room temperature, dry, undamaged | Delivery interface | | Low-retention tips | Optional, polypropylene with low-binding surface | Same as above | Viscous or protein-rich liquids | | Sample or reagent | Aqueous buffer, DNA or RNA solution, cell suspension, solvent | Equilibrated to room temperature unless the protocol specifies otherwise | The liquid you measure | | Distilled or deionized water | Reagent grade | Room temperature | Pre-wetting when you do not want to consume sample, and gravimetric checks | | Waste vessel | Beaker or reservoir designated for discarded pre-wet volumes | Labeled,化学waste segregated as appropriate | Receiving discarded draws | | 70 percent ethanol | Ethanol in water, v/v | 70 percent | Wiping the pipette body and cone, flammable | | Analytical balance | Calibrated, readability 0.1 mg for small volumes | Leveled, draft shield closed | Optional gravimetric verification | **Safety note.** 70 percent ethanol is flammable. Keep it away from open flames and hot surfaces, and cap the bottle when not in use. When the liquid you are pipetting is a biohazard, a volatile solvent, or a corrosive reagent, perform pre-wetting inside a biosafety cabinet or chemical fume hood as appropriate, wear the gloves and eye protection your institution requires, and discard pre-wet volumes into the correct waste stream, never back into the reagent stock. Filter tips are sharps-adjacent waste and go into the biohazard sharps stream after use with biological material. ## Pre-Wetting Pipette Tips Step by Step The following routine assumes an air-displacement pipette and a standard forward pipetting mode. Read the reason after each step so you understand what you are protecting, because understanding is what lets you adapt the routine to an unfamiliar liquid or a new pipette without guessing. 1. **Set the volume within the reliable portion of the range.** Stay between roughly 25 and 100 percent of the pipette's nominal maximum whenever the protocol allows. Reasons: a pipette's specified accuracy and precision degrade at the bottom of its range, and small volumes suffer the largest relative first-draw losses. If you need 8 microliters routinely, use a 2 to 20 microliter pipette, not a 20 to 200 microliter pipette. 2. **Equilibrate liquids, tips, and pipette to the same temperature.** Take reagents and a tip box out of the cold 20 to 30 minutes before pipetting, or pre-warm refrigerated buffer in your hands or a water bath if the reagent tolerates it. Reason: temperature differences between the tip, the trapped air column, and the liquid cause the aspirated volume to expand or contract and can drive meniscus drift after you lift the tip out. If the protocol requires keeping reagents on ice, pre-wet with the cold liquid itself, accept the residual temperature effect, and be consistent about it across all standards and samples. 3. **Mount the tip with a firm press and a slight twist.** Press the pipette shaft into the tip while giving a small rotational push, and check the seal visually. Reason: a seated tip forms an airtight seal. A partially seated tip leaks air during aspiration, which shows up as an aspirate that looks short or bubbles in the tip, and no amount of pre-wetting corrects a leaking seal. 4. **Aspirate the set volume and dispense it to waste, twice.** Press the plunger to the first stop, immerse the tip 2 to 3 millimeters into the liquid, release the plunger smoothly, hold for about one second, then dispense into a waste vessel by pressing to the first stop and then the second stop to blow out. Repeat once. Reason: the first draw saturates the dead air with vapor and leaves a conditioning film on the plastic, the second draw confirms the tip is delivering reproducibly and flushes any manufacturing residue. Two cycles are the standard practice for aqueous and most routine liquids. 5. **Choose the pre-wet liquid deliberately.** For a precious sample or a dilution series, pre-wet with water or with the buffer the sample is dissolved in, then change to a fresh tip for the actual draw if carryover is a concern, or pre-wet with the liquid you will discard the first draw of into waste, never back into stock. Reason: whatever liquid is left in the tip from pre-wetting ends up in the next draw. Pre-wetting a 10 microliter tip with concentrated stock and then drawing your first standard adds a small, unmeasured slug of stock to that standard. 6. **Aspirate the measured volume with a smooth, slow plunger release.** After pre-wetting, press to the first stop, immerse the tip 2 to 3 millimeters below the meniscus, release the plunger at a controlled speed over about one second, pause one second, then withdraw the tip vertically from the liquid, sliding along the vessel wall if you need to shed an external droplet. Reason: fast plunger release causes splashing, bubbles, and, with viscous liquids, under-aspiration because the liquid cannot flow in fast enough to follow the piston. The pause gives the meniscus time to settle before the tip leaves the liquid. 7. **Dispense against the wall of the receiving vessel.** Touch the tip to the side wall at a shallow angle, press smoothly to the first stop, pause one second, then press to the second stop to blow out the residual film, and only then withdraw the tip while holding the second stop. Reason: the wall gives the expelled liquid a surface to run down, the pause lets the film drain, and the blow-out recovers the volume retained in the tip. Then wipe or touch off the external droplet on the wall before moving on, so you do not carry liquid to the next well. 8. **Keep the rhythm identical across all your standards, samples, and replicates.** Use the same immersion depth, the same aspiration and dispensing speed, the same one-second pauses, and the same pre-wet cycle count for every condition in the experiment. Reason: consistent technique converts the residual systematic errors into biases that cancel between standards and samples, while inconsistent technique converts them into noise and differential bias that do not cancel. 9. **Change tips when the liquid changes.** Use a fresh tip for each new reagent, each concentration of a standard series, and each biological sample. Reason: the conditioning film that makes pre-wetting work also holds a small amount of the previous liquid, which is exactly what you do not want to transfer into your next standard or your next sample. 10. **For multi-channel pipettes, verify every tip is seated before you start.** Press the pipette head onto the tip rack with even pressure across the full row, and check alignment visually against the rack. Reason: a single poorly seated tip on an eight-channel head silently produces one bad column of data, and multi-channel work has its own systematic error sources that disciplined handling addresses, as shown in a study that optimized multi-channel pipettor use specifically to improve kinase assay inhibition data [4]. ## What Good Pre-Wetting Looks Like: Expected Results and How to Read Them ![Worked calculation comparing the mean mass of ten pre-wetted and ten dry 100 microliter water deliveries.](/article-images/illustrations/pre-wetting-pipette-tips-gravimetric-bias.webp) You cannot see a microliter of retained film. You read the effect of pre-wetting through the behavior of your data and through a few direct checks. **Direct checks during setup.** After pre-wetting and drawing the measured volume, the meniscus inside the tip should be concave, clean, and stable, with no bubbles clinging to the walls and no liquid creeping up or down while you hold the pipette still. With water and a correctly functioning pipette, the visible meniscus height in the tip should look the same for consecutive draws. If draw one, two, and three visibly differ before pre-wetting and converge after it, you have just watched the effect directly. **Repeatability of delivered volumes.** The practical expectation for careful manual technique is replicate deliveries that agree within a percent or two at mid-range volumes, and somewhat wider scatter at the bottom of the pipette's range. Validation work on automated systems gives a useful reference point for what engineered, hands-free precision looks like, with about 2 percent technical imprecision in a cell survival assay and 4.5 to 6 percent in bioluminescent and ELISA assays [3]. Manual work that approaches this range is performing well. **Consistency across an experiment.** In a standard curve, the linear regression of signal against concentration should have little scatter around the fitted line. In a plate-based assay, replicate wells should not show a positional pattern, for example all row A wells reading high, because such patterns usually trace back to handling differences such as uneven tip seating or evaporative exposure rather than biology. When intraday variation across wells of the same treatment group exceeds the technical imprecision of the measurement itself, as documented in the automated seeding and exposure work cited above, the excess is biological and positional variation, and consistent pipetting is what keeps the technical contribution from adding to it [3]. **Gravimetric verification.** If you want to measure the effect rather than infer it, deliver ten pre-wetted 100 microliter volumes of water onto a calibrated analytical balance and compare the mean mass to the expected mass, then repeat with ten dry-tip deliveries. Water at room temperature has a density near 1.00 milligram per microliter, so mass in milligrams approximates volume in microliters after the small buoyancy correction your calibration service applies. The difference between the two means is your first-draw bias, measured. This is the same gravimetric logic professional pipette calibration uses. ## Pipette Calibration: What Pre-Wetting Can and Cannot Fix Pre-wetting corrects errors that originate at the tip. It does nothing for errors that originate in the instrument. A pipette whose piston seal has worn, whose piston travel has drifted, or whose display no longer matches delivered volume will produce consistently wrong volumes whether or not you pre-wet. The two error sources are complementary, and a competent pipetting technique addresses both. Pipette calibration is the periodic gravimetric verification of a pipette's delivered volumes, typically performed by aspirating and dispensing water onto a calibrated analytical balance at several volumes across the range, then comparing the mean and scatter against the manufacturer's tolerance for that volume. Under the international standard for piston-operated volumetric apparatus, ISO 8655, calibration is done under controlled conditions of temperature and humidity with an appropriately precise balance, and the number of test volumes and repetitions follows the standard's procedure. Keep the specifics qualitative in your own practice and defer the exact tolerances to the manufacturer's specification sheet for your model, because tolerances differ by brand, range, and single versus multi-channel design. Three practical rules connect calibration to pre-wetting. 1. **Calibrate on a schedule.** Most laboratories verify pipettes every 6 to 12 months, and always after a drop, a suspected leak, or any event that could have deformed the shaft or piston. Your institution's quality system sets the required interval. 2. **Pre-wet during calibration checks too.** A gravimetric calibration performed with dry tips measures a mixture of instrument error and first-draw bias. Calibrate the way you work. If your SOP requires pre-wetting, the calibration should include pre-wetting so the instrument is verified under operating conditions. 3. **Pre-wetting is not a substitute for repair.** If a pipette fails gravimetric verification, send it for service. No pipetting technique compensates for a mechanical fault, and a study that specifically optimized multi-channel pipettor handling to improve assay data did so against a backdrop of a functioning, verified instrument [4]. The instrument defines the ceiling, and technique determines how close you get to it. ## Troubleshooting | Symptom | Likely cause | Fix | |--|--|--| | First delivered volume consistently low, later deliveries fine | Dry-tip evaporation and retention on the first draw | Pre-wet twice before the measured draw, and do it for every tip in the series | | Bubbles form in the tip during aspiration | Plunger released too fast, tip immersed too deep, tip seal leaking, foamy liquid | Slow the plunger release, immerse 2 to 3 millimeters only, reseat the tip, pre-wet slowly for foam-prone liquids such as detergent-containing buffers and protein solutions | | Liquid droplet hangs on the tip orifice after dispensing | Residual film plus static, or the blow-out was skipped | Always dispense to the second stop, touch the droplet off on the vessel wall, and use low-retention tips for surfactant or protein-rich liquids | | Delivered volumes vary from draw to draw | Inconsistent immersion depth or rhythm, liquid on the outside of the tip being carried in or out, pipette held at a steep angle | Standardize immersion depth and timing, touch off external droplets, keep the pipette within about 20 degrees of vertical during aspiration | | One column of a 96-well plate is an outlier | One tip on a multi-channel head is not seated or is damaged | Reseat the full tip row with even pressure and inspect tips before starting, an error source specifically relevant to multi-channel optimization work [4] | | Volumes drift low over a long session | Evaporation from the reservoir, tip and liquid temperatures diverging, operator fatigue changing rhythm | Cover the reservoir between draws, re-equilibrate, take a break, and re-pre-wet whenever conditions change | | Volume looks correct but assay standards are systematically off | Pre-wetted standards pipetted against dry-tip samples, or carryover from pre-wetting with concentrated stock | Apply the identical pre-wet routine to every condition, and pre-wet with a discardable liquid rather than concentrated stock | | Dripping from the tip while holding the pipette | Worn seal or piston, cracked tip cone, mismatched tip brand | Test with a fresh compatible tip, then send the pipette for calibration or service, since mechanical faults are not correctable by technique | ## Variations for Specific Liquid Types ![Decision path asking about volume, liquid type and accuracy needs to say whether to pre-wet, reverse pipette or skip.](/article-images/illustrations/pre-wetting-pipette-tips-prewet-or-skip.webp) ### Viscous liquids Glycerol, concentrated protein solutions, lysis buffers with high glycerol content, and enzyme stocks flow slowly. Aspirate slowly, release the plunger slowly, and consider reverse pipetting. In reverse mode you press the plunger to the second stop before aspirating, draw the liquid, and dispense by pressing only to the first stop, which delivers the set volume while leaving the excess and the conditioning film in the tip. Reverse pipetting is deliberately a residual-in-tip method, which makes it the natural extension of the pre-wetting principle for viscous and foamy liquids. Pre-wet with the same liquid before reverse-aspirating the measured volume, and discard the tip with its retained excess afterward. ### Volatile liquids and hot or cold samples Ethanol, acetone, and other volatile solvents evaporate inside the tip fastest when the tip is dry, so pre-wetting gives the largest proportional benefit here. Work quickly, keep the reagent bottle capped, and consider keeping tips and liquid at the same temperature. For samples on ice, pre-wet with the cold liquid itself so the measured draw happens with a thermally equilibrated tip, and apply the same treatment to every condition in the experiment. ### Small volumes below 10 microliters At 2 microliters, retention and evaporation losses that are negligible at 200 microliters become a meaningful fraction of the target. Pre-wet before every draw, not just the first in a series, because at this scale each draw re-establishes equilibrium. Use a pipette sized so the volume is at least a quarter of its range, use low-retention tips, and pre-wet with the actual liquid only when carryover into stock is not a concern, otherwise pre-wet with buffer or water and use a fresh tip for the measured draw. ### Conditioning tips that carry an extraction phase A useful illustration of the same principle at industrial scale is disposable pipette extraction, or DPX, in which the tip itself carries a sorbent phase and liquid is cycled through it repeatedly. Optimized DPX methods use multiple fixed-time cycles, for example three 30-second extraction cycles in a cork biosorbent method for lake water contaminants [5], two extraction cycles and five desorption cycles in a nitrosamine method for pharmaceuticals that achieved recovery up to 98 percent and precision and accuracy below 15 percent [6], and a combined protein precipitation and zirconia-sorbent DPX workflow for plasma cannabinoids that reached lower limits of quantification of 0.1 nanogram per milliliter for anandamide and 2-arachidonoylglycerol and 1 nanogram per milliliter for cannabidiol and tetrahydrocannabinol [7]. The repetition in those cycles serves exactly the same purpose as your two pre-wet draws: it brings the tip's interior into a reproducible equilibrium with the liquid before any measurement is taken. If your work ever moves from plain liquid handling to sorbent-loaded tips, cycle count and cycle time become pre-wetting-equivalent parameters to standardize. ### Automated and semi-automated liquid handling Robotic pipetting systems do not relax the underlying requirements, they enforce them. Before any automated system was used for toxicity studies, the researchers validated pipetting accuracy first and only then compared automated seeding homogeneity against expert technicians, finding intraday biological variation larger than the technical variation, which had to be accepted as inherent to the plate format [3]. The lesson for manual work is the ordering. Validate your own technique, including your pre-wetting routine, before you trust the data it produces, and do not confuse biological well-to-well variation with pipetting error when you troubleshoot a plate. ## Storage and Stability Notes Pipette tips are consumables with long shelf stability when stored correctly. Keep unopened tip boxes sealed in their original packaging, stored away from direct sunlight, in a dry cabinet at room temperature, and use them before the printed expiration date if one is printed. Sterility, not performance, is what expires first on most tip boxes, and irradiated tips slowly lose their sterility guarantee even though their geometric dimensions are essentially stable. Once a box is opened, close the lid after every use to limit dust, particulates, and, in a shared lab, aerosol contamination of the rim and orifice of the remaining tips. Do not autoclave tips that the manufacturer has already sterilized unless the manufacturer states the tips tolerate re-sterilization, because repeated autoclaving can warp the orifice and change retention behavior. Low-retention tips and filter tips have no special storage requirements beyond the above, but filter tips should be discarded rather than re-wetted and reused after contact with samples. The pre-wetted tip itself is not a stable state. A tip you pre-wetted thirty seconds ago is not equivalent to one you pre-wetted ten minutes ago, because the conditioning film partially dries and the trapped air can lose its vapor saturation, particularly with volatile liquids. Pre-wet immediately before the measured draw. If a pipetting session is interrupted, for example by a stopped centrifuge run, re-pre-wet the tip or take a fresh one and re-establish your routine before resuming measured draws. ## Common Mistakes and Limitations The most common mistakes with pre-wetting pipette tips are failures of consistency rather than failures of understanding. Each item below names the mistake, the consequence, and the correction. 1. **Pre-wetting some conditions but not others.** This converts a uniform first-draw bias into a differential bias between groups. If you pre-wet your standards but pipette your unknown samples dry, every calculated concentration is skewed. Correct by writing the pre-wet requirement into the SOP and applying it to every aspiration that contributes to a measurement. 2. **Pre-wetting directly from concentrated stock.** The residual film of stock left in the tip after the pre-wet draw is dispensed into your first standard, adding an unmeasured slug of analyte. Correct by pre-wetting with water, buffer, or a deliberate sacrificial aliquot in a separate vessel, and never returning pre-wet volumes to the stock bottle. 3. **Using the same pre-wetted tip for multiple standard concentrations.** The film carries the previous concentration into the next draw, which flattens the top of a standard curve and compresses your dynamic range. Correct with a fresh tip per concentration, which also satisfies good carryover practice. 4. **Inconsistent cycle counts.** One draw for some tips, three for others, whatever the operator's mood dictates. The retained film volume reaches a stable state after one to two cycles, and deliveries before stabilization differ from deliveries after. Correct by fixing the number, usually two, in your pipetting technique and applying it identically everywhere. 5. **Pre-wetting with liquid at a different temperature than the measured draw.** Pre-wetting with room-temperature water and then drawing a cold sample leaves a thermally conditioned air column above a cold liquid, which distorts the meniscus. Correct by pre-wetting with the liquid you intend to measure, at the temperature you intend to measure it at, whenever carryover permits. 6. **Skipping pre-wetting because the volume is large.** At 1 milliliter the relative first-draw error is small, and skipping is defensible for rough transfers. The mistake is carrying that logic backward and skipping at 20 microliters, where the same absolute loss is proportionally large. Correct by scaling your diligence to the relative error, not the absolute volume. 7. **Treating pre-wetting as a fix for mechanical problems.** A leaking seal, a worn piston, or a bent shaft causes errors that pre-wetting cannot touch. The limitation to state plainly is that pre-wetting addresses tip-level effects only. Instrument-level accuracy comes from pipette calibration and maintenance, and the two must both be in place for careful technique to matter. 8. **Ignoring tip quality.** Damaged orifices, burrs from cheap molds, and hydrophobic variation between tip brands change retention behavior. If your pre-wetted volumes still scatter more than expected, swap tip brands or move to low-retention tips before blaming your hands. 9. **Assuming pre-wetting eliminates the need for good pipetting technique.** Immersion depth, plunger speed, verticality, wall dispensing, and dwell times all still apply. Pre-wetting removes one systematic error and leaves the others intact. 10. **Carrying the idea to the wrong conclusion with sorbent tips.** Tips designed for DPX-type extraction work need their own conditioning cycles specified by the method, and treating them like plain tips, or pre-wetting them with the wrong solvent, ruins the extraction phase [5][7][6]. Read the method and follow its cycle specification. ## Frequently Asked Questions ### What does pre-wetting a pipette tip actually do? It saturates the air inside the tip with vapor and coats the inner wall with a thin liquid film, so the first measured draw behaves like every subsequent draw instead of losing volume to evaporation and retention. ### How many times should you pre-wet a tip? Twice for most aqueous liquids at routine volumes. Use three or more cycles for volatile solvents and very small volumes, and use a fresh tip if the liquid is precious enough that even a discardable draw is costly. ### Do you need to pre-wet tips for every liquid type? No. Aqueous buffers at volumes above roughly 100 microliters gain little from pre-wetting in rough transfers, while volatile solvents, viscous liquids, and any volume below about 20 microliters gain the most. ### Does pre-wetting matter below 10 microliters? Yes, more than anywhere else. At 2 microliters a fixed retention and evaporation loss is a large fraction of the target volume, so pre-wet before every measured draw. ### Should you pre-wet when making a standard curve? Yes, and you must do it identically for every standard and every unknown. Uneven pre-wetting between standards and samples introduces a differential bias that distorts calculated concentrations. ### What is reverse pipetting and when is it better? It is a mode where you aspirate by pressing to the second stop and dispense by pressing only to the first stop, leaving excess liquid in the tip. It is better for viscous and foamy liquids because it compensates for their tendency to cling to and under-fill the tip. ### Does pre-wetting waste reagent? It uses one or two extra tip-volumes per tip, typically a few hundred microliters per session. Use water, buffer, or a designated sacrificial aliquot to keep the cost negligible, and never discard pre-wet volumes back into stock. ### How does pre-wetting relate to pipette calibration? They address different error sources. Calibration verifies and corrects the instrument, pre-wetting corrects the tip, and both must be current for accurate volumes. Pre-wet during gravimetric checks so the calibration reflects the way you actually work. ## Sources 1. [Comparative evaluation of dimensional accuracy of resultant casts obtained from different impression techniques: An in vitro study](https://doi.org/10.4103/injo.injo_12_23) 2. [Optical three-dimensional scanning acquisition of the position of osseointegrated implants: an in vitro study to determine method accuracy and operational feasibility.](https://pubmed.ncbi.nlm.nih.gov/18657149/) 3. [Validation of automated pipetting systems for cell culture seeding, exposure and bio-analytical assays in sulfur mustard toxicology.](https://pubmed.ncbi.nlm.nih.gov/31809884/) 4. [Maximizing the Precision and Accuracy of a Multi-Channel Pipettor to Optimize Inhibition Data from a Kinase Assay](https://www.semanticscholar.org/paper/e7fec015a8e15f877cb51f9d15bb3ed68a1b969b) 5. [A natural and renewable biosorbent phase as a low-cost approach in disposable pipette extraction technique for the determination of emerging contaminants in lake water samples.](https://pubmed.ncbi.nlm.nih.gov/30667178/) 6. [Development of a reliable method for determination of N-nitrosamines in medicines using disposable pipette extraction and HPLC-MS analysis.](https://pubmed.ncbi.nlm.nih.gov/38747210/) 7. [A Disposable Pipette Extraction-UHPLC-MS/MS Method Based on Removal of Phospholipids to Determine Anandamide, 2-Arachidonoylglycerol, Cannabidiol, and Δ9-Tetrahydrocannabidiol in Plasma Samples.](https://pubmed.ncbi.nlm.nih.gov/39806770/)