It can be a challenge to transfer large volumes of liquid quickly. To improve speed, be sure to use 5ml pipettes (or 1ml pipettes at the least) to transfer liquid from the vacutainer to the tubes or plates. Many labs choose to manually perform these preprocessing steps of centrifuging and pipetting. Plasma to be used for liquid biopsy is generally prepared using the double centrifugation protocol. This ensures a clean capture of the plasma that is free of contaminating cellular DNA that is typically higher molecular weight. While an instrument could be used to prepare the plasma, it requires expensive and complex camera systems to inform the pipette where the layers are. Without a camera system, there is no way to know where the plasma level starts, which could result in cellular contamination. However, if you have enough sample, you could pipette from the top at a fixed volume. For example, if 2ml of plasma is enough, there should be sufficient sample in a 10ml blood collection that a fixed volume far from the border of the layers could be used without disrupting the cellular fraction post-centrifugation.
Digestion and Lysis
The next step where the volume and viscosity of the samples plays a role is in the digestion and lysis step. Although mixing large volumes of a viscous solution can pose some challenges, they can be easily overcome with proper equipment. We provide a large volume Heater Shaker Magnet (HSM) module for this purpose that can be adapted to all major liquid handlers or used separately.
The binding step is the most universally challenging because the volumes can become quite large. For example, 3ml of plasma will typically require 1–3 volumes of binding buffer (depending on the supplier and kit), leading to a total volume of 6–12ml that requires mixing. This step requires extra caution as insufficient mixing is one of the leading causes of low yield. Visually inspect the sample to ensure the beads are fully suspended during the binding and that a vortex fully forms, suggesting thorough mixing. If these visual parameters are met, you can be confident that binding is occurring with sufficient time. Confirmation can be made by further processing the sample manually to avoid the risk of other steps influencing the result. For more information on experimental design and troubleshooting, download the "Automating Nucleic Acid Extraction — A How-to Experimental Guide".
There are a variety of methods to complete the binding step:
- Using a Promega Heat Shaker Magnet (HSM) or rotisserie (most efficient)
- Sequential binding using a KingFisher™ Presto Purification System (Thermo Fisher Scientific), a particle mover that can be integrated onto most liquid Handlers (e.g., Hamilton or Tecan workstation)
- Sequential binding on a liquid handler (least efficient)
The most efficient method is to use the HSM and 5ml pipettes, or the Presto System. If using the HSM, it can be helpful to add the magnetic particles while the liquid is shaking to keep them suspended. Some particles settle rapidly and can be difficult to resuspend in viscous solutions full of proteins that could cause bead aggregation. When using the HSM approach, the binding itself is very efficient, but the liquid removal step and bead transfer is less efficient compared to the Presto System. The Presto System supports a lower maximum volume and therefore requires sequential binding, but it gains speed during the transfer steps. Sequential binding on a liquid handler is the least effective. Unless you are working with low volumes, the number of sequential binding steps makes it extremely slow.
Following this binding (mixing) step, the samples will need to be transferred from large tubes to a more convenient format, such as a 96 well plate. This requires magnetizing the beads and removing the solution, followed by resuspension of the particles and then transferring. This tends to be a very slow process as the instrument will need to aspirate multiple times per tube before transferring the beads.
During the wash steps, magnetic particles can clump due to the quantity of protein being inadvertently concentrated from the large sample volume. This is especially true during the first wash. The best way to track your progress is to visually verify that the clump of particles is disrupted, otherwise your eluates will be impure and possibly inhibited downstream.
To learn more about overcoming this problem, read the "Automating Nucleic Acid Extraction" guide, which walks through the protocol for automating nucleic acid extraction. You will find a detailed guide including video instruction, experimental design and troubleshooting tips to support your method development.
Need help with automation? Partner with our Field Support Scientists, who have decades of experience automating our kit chemistries on a wide range of platforms and throughput scales. We’ll help you customize the scale, application, and chemistry exactly to your needs, regardless of platform.
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