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Beckman Coulter AMPure XP beads protocol for PCR purification
Automatic magnetic bead purification with ASSIST PLUS pipetting robot
Agencourt AMPure XP beads (Beckman Coulter) are used for DNA purification in a variety of applications, including PCR, NGS, cloning and microarrays. The ASSIST PLUS pipetting robot provides a solution for optimal bead separation and maximized recovery of precious samples. User guidance throughout the entire protocol ensures an error-free pipetting procedure. Careful and accurate handling of the magnetic beads by the ASSIST PLUS leads to superior reproducibility and consistency during the experiment. Taken together, the ASSIST PLUS provides researchers with an easy and highly efficient way to purify DNA from PCR reactions using AMPure XP magnetic beads.
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Table of contents
Agencourt AMPure XP beads (Beckman Coulter) are used for DNA purification in a variety of applications, including PCR, NGS, cloning and microarrays. The ASSIST PLUS pipetting robot provides a solution for optimal bead separation and maximized recovery of precious samples. User guidance throughout the entire protocol ensures an error-free pipetting procedure. Careful and accurate handling of the magnetic beads by the ASSIST PLUS leads to superior reproducibility and consistency during the experiment. Taken together, the ASSIST PLUS provides researchers with an easy and highly efficient way to purify DNA from PCR reactions using AMPure XP magnetic beads.
Key benefits
- The VIAFLO and VOYAGER electronic pipettes, in combination with ASSIST PLUS, provide unmatched pipetting ergonomics.
- Optimal pipette settings, including tip immersion depth, pipetting speeds and angles, maximize reproducibility and sample recovery.
- Exact positioning of the pipette tips in the sample wells avoids the risk of disturbing the ring of magnetic beads or bead carryover.
- The ASSIST PLUS automates many steps of a magnetic bead purification protocol and guides the user through the remaining manual operations to ensure an error-free process.
Overview: How to automate PCR purification steps with ASSIST PLUS
The ASSIST PLUS is used to purify DNA samples using AMPure XP beads (Beckman Coulter). The pipetting robot runs a VOYAGER 8 channel 125 μl electronic pipette with 125 μl Sterile, Filter, Low Retention GRIPTIPS®. The use of Low Retention GRIPTIPS guarantees optimal liquid handling of viscous (AMPure XP buffer) and volatile (70 % ethanol) solutions.
Below is an example set-up for 24 samples, preparing 10 µl DNA samples (position B) with 18 µl of AMPure XP beads (position A). The pipetting programs were prepared according to the manufacturer’s protocol (AMPure XP, Beckman Coulter) using VIALAB software.
The protocol is divided into two programs that guide the user through every step of the PCR purification process.
- Program 1: Binding (AMP_BINDING)
- Program 2: Washing and elution (AMP_WASH_ELUTE)
Downloads: AMPure XP beads protocol and reports for automated PCR
Here you can download the complete protocols and reports used in this application and use them on your ASSIST PLUS. The protocol can be customized with the VIALAB software.
Experimental set-up: Program 1
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Deck position A: |
PCR 8 tube strip containing the AMPure XP beads (Figure 1, blue), placed onto a cooling block from INTEGRA. Note: the cooling block is just used as a support in this instance, and not for cooling down the samples. |
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Deck position B: |
96 well plate with 24 DNA samples for purification (Figure 1, green). |
| Deck position C: |
96 well ring magnet. |
Run program 1: Transfer & Binding
Select and run the AMP_BINDING program on the VOYAGER electronic pipette. The ASSIST PLUS pipetting robot immediately starts the protocol.
1. AMPure XP transfer
Transferring AMPure XP beads from an 8 tube PCR strip to a 96 well plate containing the DNA samples.
To ensure the AMPure XP buffer is homogenous, the beads are resuspended by pipetting up and down 10 times before being transferred to the samples. The beads and DNA fragments are thoroughly mixed together before the pipette automatically starts the timer for 5 min. incubation, ensuring optimal conditions for the DNA strands to bind onto the magnetic beads.
Tip: Using Low Retention GRIPTIPS rather than regular GRIPTIPS prevents the loss of AMPure XP beads during the pipetting steps (see Figure 2).
2. Magnetic separation of the AMPure XP beads
Separating the magnetic beads from the PCR samples.
A message instructs the user to move the plate (position B) onto the magnet (position C). Continue the program to start the timer. After a two minute incubation on the magnet the beads form a ring in the sample well and the solution becomes clear. The program resumes automatically, and the supernatant is removed. On completion of this step, the pipette prompts the user to continue with the AMP_WASH_ELUTE program and to replace the labware on position A with the 8 row polypropylene (PP) reagent reservoir containing the ethanol and elution buffer.
Tip: The supernatant is aspirated slowly using the Tip Travel feature of the ASSIST PLUS to avoid disturbing the ring of beads. The Tip Travel feature keeps the tip immersion depth constant during aspiration and dispensing. 5 μl of supernatant remain in the plate to prevent beads being drawn out during aspiration.
Experimental set-up: Program 2
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Deck position A: |
The 96 well PCR cooling block is replaced by an 8 row polypropylene (PP) reagent reservoir filled with 70 % ethanol in row 1 (blue) and elution buffer in row 2 (orange). Row 8 is used for waste (purple). |
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Deck position B: |
Emtpy 96 well plate. |
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Deck position C: |
96 well ring magnet and 96 well plate with 24 DNA samples for purification (green). |
Run program 2: Washing & Elution
Start the AMP_WASH_ELUTE program on the VOYAGER electronic pipette. The ASSIST PLUS washes the beads twice by automatically adding and removing ethanol.
3. Magnetic bead clean-up
Washing the magnetic beads twice with 70 % ethanol.
The programmed pipette settings allow the beads to be washed without disturbing the bead ring. At the end of the second washing step, all the ethanol is removed. If necessary, an additional drying time can easily be added using VIALAB software.
Tip: The use of Low Retention GRIPTIPS prevents ethanol from dripping while traveling from position A to position C (see Figure 6).
4. Elute samples from the magnetic beads
Eluting the samples from the magnetic beads by adding an elution buffer.
The pipette prompts the user to move the reaction plate from the magnet (position C) to position B. Continuing the protocol, the ASSIST PLUS transfers the elution buffer to the DNA samples bound to the magnetic beads (position B, orange). After mixing carefully and thoroughly 10 times, the pipette prompts the user to place the 96 well plate on the magnet (position C).
5. Transfer the sample eluates
Transferring the sample eluates into a new 96 well plate.
As indicated by the pipette, place a new 96 well plate onto position B and continue the program. The sample eluates are then transferred into the new plate automatically.
Tip: Optimized pipette settings (aspiration speed, volume, height, tip travel and tip touch) allow the volume of eluate transferred to be maximized without carryover of beads (see Figure 6). A tip touch after the transfer removes droplets that may still cling to the end of the pipette tips. Pipetting heights on the ASSIST PLUS can be fine-tuned at any time. Performing a test run with water before implementing any new assay is an ideal way to optimize pipette settings.
Results
Conclusion
- Magnetic bead purifications can be easily automated on the ASSIST PLUS pipetting robot.
- Optimized tip immersion and pipette settings together with the use of Low Retention GRIPTIPS® allow maximum sample recovery at the end of the purification protocol.
- The pipette loaded onto the ASSIST PLUS prompts the user when needed, eliminating the risk of human errors.
- VIALAB programs can be easily adapted to specific labware.
- Prolonged pipetting tasks lead to repetitive strain injury. This can be avoided by automating these steps with the ASSIST PLUS.
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Instruments and accessories
ASSIST PLUS, Pipetting Robot
INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:
- automates pipetting tasks,
- eliminates physical strain and
- ensures superior reproducibility and
- error free pipetting.
Part No. 4505
VOYAGER, 8 Channel, 125 µl
VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.
- Single handed operation leaves the other hand free to handle labware.
- On the fly access to up to 3 user-defined tip spacings.
- These user defined tip spacings are saved. No need to memorize spacings of different labware formats.
Part No. 4722
GRIPTIPS®, 125 µl, Sterile, Filter, Low retention
INTEGRA uses a unique polypropylene blend to mold Low Retention GRIPTIPS® with heightened hydrophobic properties. Using Low Retention GRIPTIPS® prevents low surface tension samples from spreading out and “wetting” the inner wall of the tips, allowing them to bead-up for a maximum liquid recovery.
Part No. 6565
PCR 96 well cooling block
Aluminium 96 and 384 well PCR cooling blocks that keep reagents chilled during sample preparation in a PCR plate. The cooling blocks feature a SBS footprint.
Part No. 6250
8 Row Polypropylene Reservoirs (6371)
The polypropylene design improves the reservoirs chemical compatibility and also allows the reservoirs to be autoclaved.
- Reagent reservoirs for 96 well plates
- Pyramid bottom
- Each row holds 32 ml
Part No. 6371
BECKMAN COULTER Life Sciences: Agencourt AMPure XP
A highly efficient, easily automated PCR purification system that delivers superior quality DNA with no salt carryover. Requiring no centrifugation or filtration, Agencourt AMPure XP can be easily used in manual and automated 96- or 384-well formats.
Source: Website Beckman Coulter
ALPAQUA Accelerating Genomic Discovery: 96S Super Magnet Plate
SBS-compliant ring magnet plate with extra strong ring magnets ideal for large volume separations. Compatible with many 96-well PCR plates, round bottom deep well and half deep well plates.
Source: Website Alpaqua Engineering
Greiner Bio-One: 96 Well Polypropylene Microplates
SAPPHIRE MICROPLATE, 96 WELL, PP, FOR RT PCR, WHITE, HALF-SKIRT, FOR LIGHT CYCLER, 10 PCS./BOX
Source: Website Greiner Bio One
CORNING: Corning® Thermowell ® GOLD 0.2 ml 8-Well PCR tube strips
- Tube strips consist of eight 0.2 mL tubes connected together
- Cap strips are sold separately
- Made of thin wall polypropylene and designed for precise fit in heat blocks for optimum heat transfer
- Certified DNase- and RNase-free
- Autoclavable at 121°C
Source: Website Merck KGaA
Download
Download this App Note as PDF file
DownloadBeckman Coulter AMPure XP beads protocol for PCR purificationCustomer’s voice
Our lab loves the INTEGRA ASSIST PLUS. Equipment always functions well and accompanying software is the most intuitive and user-friendly that we have encountered. The accompanying pipettes can be used at the bench as well as on the ASSIST PLUS which increased the value of the entire system for our lab. The automated reformatting between 96-well plates, 384-well plates, and varied sizes of tubes have made this product well worth the cost. We love the ASSIST PLUS and are looking to buy another one. (Review published on Select Science)
