What is dead volume?
Dead volume refers to the residual volume of liquid left in the well or lost to waste that cannot be used in the application due to the risk of aspirating air. This can be due to well geometry and a number of other factors. High dead volumes may be concerning when pipetting valuable liquids such as master mixes, enzymes, or antibody solutions but may be acceptable in the case of low cost reagents like water or sodium solutions. Since we are no longer pipetting by hand, we need to also consider that we are only able to access the wells from the top down rather than having a full range of motion to maneuver and consolidate small droplets within the wells of labware, as is the case with most liquid handling platforms.
What factors contribute to dead volume?
The amount of dead volume depends on a few factors. These can be attributed to the labware type and geometry, the properties of the liquid used, the pipette being used, as well as your specific environmental conditions..
The geometry and material of your labware can greatly affect the dead volumes required. 96 well plates tend to have a conical shape, with a reduced surface area as you approach the bottom of the labware. This geometry is ideal for reducing dead volumes where needed- conical geometry with decreasing surface area toward the bottom will allow for smaller dead volumes. However, the opposite is true for things like large reagent reservoirs. The large surface area of the wells in this type of labware lend themselves to the requirement of a larger dead volume. When choosing labware in the interest of reducing dead volumes, look for items with conical or V shaped bottoms. If you are not worried about high dead volumes, flat bottom plates are perfectly acceptable!
Material also plays a role in dead volumes. Most scientific labware is manufactured from materials that are by default hydrophobic and tend to bead liquids, reducing liquid adhesion to the walls of the well and reducing the dead volumes by wicking the liquids to the bottom of the well where it can be easily accessed. Some hydrophilic materials and coatings do exist however, usually in the case of plates intended for cell culturing and protein binding. Both of these plate material options are perfectly acceptable for use on the OT-2, but bear in mind the effects on dead volume and the goals of your workflow when selecting labware.
Liquids have many properties that may affect required dead volumes. The largest of these are threefold: viscosity, surface tension, and pipette tip submerge depth.
Viscous liquids will need to be aspirated and dispensed at a lower flow rate to ensure accurate volumes and a full dispense of the liquid. Quickly aspirating viscous liquids can lead to inaccurate pipetting volumes and you may run out of liquid before expected due to this.
Surface tension refers to the property of a liquid that allows it to resist some external force, like a pipette tip plunging through the surface. Liquids with high surface tension may tend to require deeper submersion of the pipette tip in order to properly aspirate without introducing air into the tip. This may lead to an increase in dead volumes due to the tendency of liquids to adhere to the outer wall of pipette tips.
When working with flat bottom labware, it is important to note the behavior of the liquid in a low volume state. Some liquids may tend to wick to the perimeter of the wells when the reservoir is nearing depletion, whereas some may form beads near the center.
The depth at which a pipette tip is submerged into a liquid also plays a large role in determining dead volumes. The deeper a tip enters into a liquid, the more contact that liquid has with the outer surface of the pipette tip. As the tip-liquid contact surface area increases, the resulting dead volume increases. To avoid this it’s best to aspirate your liquids from just below the surface, accounting for the change in liquid volume resulting from the pipetting action.
Pipette type and technique
The type and number of channels on your pipettor also play a role in dead volumes. Multichannel pipettors of high maximum volume will tend to result in the highest dead volumes, whereas a single channel low maximum volume will result in the lowest dead volumes. This is due to the surface area the liquid is exposed to. The more surface area the liquid is in contact with, the more likely it is that some amount of liquid will adhere to the surface of the tips. Using a low maximum volume pipettor not only allows for less surface area contact, but also a more fine positioning of the tip within the well.
Some pipetting techniques can be implemented into protocols to reduce the dead volume of a protocol. Actions like blow outs, touching the tip along the destination or source wells walls, and slower aspirate and dispense speeds can help to reduce dead volumes.
Lastly, environmental conditions can impact the dead volume, these factors include air temperature and humidity.
Air temperature and the resulting surface temperature have an inverse relationship: as the air temperature increases, the surface tension decreases. Similarly, relative humidity and surface tension also have an inverse relationship. As relative humidity increases, the surface tension of the liquid decreases.
Keep these considerations in mind when designing and performing your protocols, and happy pipetting!