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How do you prevent contamination and cross-over in gradient techniques?

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Gradient techniques are widely used in laboratory settings to separate and purify different components of a mixture, such as cells, proteins, or nucleic acids. However, these techniques also pose some challenges, such as the risk of contamination and cross-over between the layers of the gradient. Contamination and cross-over can compromise the quality and integrity of your samples, leading to inaccurate results and wasted time and resources. In this article, you will learn how to prevent contamination and cross-over in gradient techniques by following some simple tips and best practices.

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Josh Preston

Research Scientist at Liverpool Heart and Chest Hospital

1 Choose the right gradient type

There are two main types of gradients: density gradients and rate-zonal gradients. Density gradients are formed by layering solutions of different densities on top of each other, creating a continuous or discontinuous gradient. Rate-zonal gradients are formed by spinning a solution containing the mixture of interest on top of a pre-formed gradient. The choice of gradient type depends on the properties and size of the components you want to separate. Density gradients are more suitable for separating components that differ in density, such as organelles or viruses, while rate-zonal gradients are more suitable for separating components that differ in size or shape, such as proteins or DNA fragments. Choosing the right gradient type can help you avoid cross-over, which occurs when the components migrate across the gradient layers and mix with each other.

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2 Prepare and handle the gradient solutions carefully

To ensure a successful separation and purification of your samples, it is essential to handle gradient solutions carefully and avoid contamination and cross-over. To do this, you should use sterile and filtered solutions and equipment, as well as compatible and stable solutions that will not react with each other or the components of interest. Additionally, be sure to use appropriate concentrations and volumes of the solutions to create a smooth and stable gradient, mixing them gently and slowly to avoid air bubbles or disrupting the gradient layers. Furthermore, store the solutions at the recommended temperature and conditions to prevent degradation or crystallization, labeling them clearly with distinct colors or containers.

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3 Load and spin the samples correctly

The loading and spinning of the samples are the final and most critical steps of the gradient technique, as any mistake or deviation can result in contamination and cross-over, ruining the separation and purification. To avoid this, use clean and compatible tubes and pipettes to load the samples onto the gradient or on top of the pre-formed gradient. Make sure to use appropriate amounts and volumes of the samples, and load them slowly and carefully to avoid splashing or disturbing the gradient layers. Additionally, use the correct speed and time for spinning according to the protocol, and stop gradually and gently to prevent shaking or breaking the tubes or gradient. Finally, use a tube cutter or syringe to collect fractions of interest without cross-contamination. By following these tips and best practices, you can ensure a high-quality and reliable separation and purification of your samples.

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Josh Preston

Research Scientist at Liverpool Heart and Chest Hospital
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In my experience isolating PBMCs, the biggest factor for improving cell yield was finding the optimal deceleration setting on the centrifuge for this particular cell type.

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4 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?

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Josh Preston

Research Scientist at Liverpool Heart and Chest Hospital
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Another key factor is streamlining the method of sample collection, as certain blood draw techniques may be more harmful to your cells than others.

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How do you prevent contamination and cross-over in gradient techniques?

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1 Choose the right gradient type

2 Prepare and handle the gradient solutions carefully

3 Load and spin the samples correctly

4 Here’s what else to consider

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