PCR purification is vital to remove unwanted compounds that could interfere with downstream applications, ensuring that only the amplicons of interest remain. Typically, this is achieved by either magnetic bead or spin column PCR purification methods. We offer solutions to support both approaches, but recommend PCR purification with magnetic beads, as these protocols are easily automated, increasing valuable walk-away time while helping to reduce errors and increase the reproducibility of your results.

How does magnetic bead purification work?

PCR purification with magnetic beads is a 3-step process involving binding, washing and elution. The liquid handling steps can be automated on benchtop platforms, eliminating the cumbersome centrifugation and vacuum manifold steps associated with spin column protocols. This improves process reproducibility, increases throughput and saves time.

Step 1: Binding

Initially, magnetic beads are added to the sample at a precise bead-to-sample ratio, typically 1.8x ratio, to effectively remove DNA fragments <100 bp. This is followed by mixing cycles and incubation to aid binding of the DNA amplicons to the beads. Magnetic separation is then used to collect the bead-bound amplicons, allowing the supernatant – containing smaller DNA fragments such as primers and primer dimers – to be easily aspirated and discarded, isolating the nucleic acids.

Step 2: Washing

Other unwanted components – unincorporated dNTPs, enzymes and salts – are washed away by adding ethanol to the sample, mixing and repeating magnetic separation. The ethanol containing the unwanted components is aspirated and discarded, and an air-drying step ensures complete evaporation of the residual solvent to prevent interference with downstream applications.

Step 3: Elution

Finally, a low-salt elution buffer or nuclease-free water is added to resuspend the dried beads and release the bound DNA – the amplicons – back into solution. Magnetic separation is then used to isolate the beads from the eluate, allowing collection of high quality, purified DNA for downstream applications.

How to streamline your magnetic bead PCR purification protocols

We offer a number of solutions to help make life in the lab as easy as possible and enhance the efficiency of magnetic bead PCR purification workflows.

• MAGFLO™ NGS magnetic beads for NGS size selection and PCR purification* – MAGFLO NGS magnetic beads offer high recovery rates of amplicons. You can order a free 1 ml sample to try them for yourself.
• MAG module – our magnetic module is a useful alternative to traditional magnetic stands. Automated vertical magnet movements mean that you no longer need to transfer tubes and plates to and from magnetic stands, eliminating the risk of a spillage. Moreover, beads are collected in one spot, rather than forming a ring-shaped pellet, making it easier to aspirate supernatants without disturbance. A variety of adapters are available for different labware types, enabling a perfect fit between the sample vessels and magnets to ensure that all the beads are captured and guarantee high yields. To boost your productivity further, the MAG module can be combined with our benchtop platforms – the VIAFLO 96 and VIAFLO 384 handheld electronic pipettes and ASSIST PLUS pipetting robot – for semi- or fully automated workflows.
• ASSIST PLUS pipetting robot – the ASSIST PLUS enables automation of our electronic multichannel pipettes and integration of the MAG module to offer fully automated, walk-away PCR purification workflows. Unlike larger liquid handling robots, this affordable solution can be set up without any prior programming knowledge. INTEGRA also provides template scripts for your convenience.
• VIAFLO 96 and VIAFLO 384 handheld electronic pipettes – semi-automate your liquid transfers with our VIAFLO 96 and VIAFLO 384 pipettes, offering simultaneous pipetting of 96 or 384 samples. In combination with a MAG module, they are ideal for high throughput, full plate magnetic bead purification workflows.

How does spin column purification work?

PCR purification with spin columns consists of binding, washing and elution steps, much like magnetic bead-based workflows. Spin columns contain a membrane with an affinity for nucleic acids and are inserted into microcentrifuge tubes. Samples are transferred into the spin column and centrifuged to isolate the DNA amplicons of a specific size. These bind to the membrane, while most unwanted components – including primers and primer dimers – pass through. Any unwanted components remaining can be removed by adding a wash buffer to the spin column, followed by centrifugation. Lastly, the elution buffer is added and the column centrifuged again to release the purified DNA amplicons of interest from the membrane.

Spin columns are available as individual columns or as 96 well silica membrane plates that can be placed on a vacuum manifold instead of being centrifuged, allowing you to adapt the workflow for higher throughput. However, the spin column method does have some drawbacks compared to magnetic bead-based purification. The membranes can get blocked easily, and the minimum elution volume is larger, resulting in lower DNA concentrations. The workflow also includes several hands-on steps with a centrifuge or a vacuum manifold, which are difficult to automate, increasing the risk of human errors.

How to streamline your spin column PCR purification protocols

A number of our liquid handling solutions can be used to streamline your spin column workflows, including:

• VOYAGER adjustable tip spacing pipettes – VOYAGER pipettes can automatically adjust tip spacing at the touch of a button, so you can easily transfer buffers from reservoirs to spin columns or silica membrane plates, eliminating the need to work with single channel pipettes.
• VIAFLO 96 and VIAFLO 384 – these 96 and 384 channel pipettes enable parallel pipetting of buffers to all 96 wells of a silica membrane plate, reducing hands-on time. Read our application note PCR product purification with QIAquick® 96 PCR Purification Kit and the VIAFLO 96 handheld electronic pipette to discover the benefits that these pipettes offer.

Learn more about our solutions to enhance your nucleic acid purification applications.

*MAGFLO NGS beads are only available in Austria, Belgium, Denmark, France, Germany, Ireland, Netherlands, Sweden, Switzerland and the United Kingdom.