PCR product purification with QIAquick® 96 PCR Purification Kit and the VIAFLO 96 handheld electronic pipette

  • PCR product purification with QIAquick® 96 PCR Purification Kit and the VIAFLO 96 handheld electronic pipette

    Semi-automated PCR product purification on the VIAFLO 96 handheld electronic pipette

    QIAquick 96 PCR Purification Kit is suitable for purifying up to 10 µg of material for downstream applications, such as sequencing, cloning, labeling and microarrays. The kit facilitates the removal of impurities like primers, unincorporated nucleotides, buffers, salts, mineral oils, agarose and enzymes. The vacuum-driven process is much faster than centrifugation, and gives high, reproducible yields. It is important to avoid cross-contamination in nucleic acid purification, and QIAGEN's column design is optimized to limit carryover of contaminants. Although QIAquick 96 provides a high throughput solution, the elution, washing and binding steps are very laborious and time consuming if performed manually. With VIAFLO 96 handheld electronic pipette, the hands-on time is reduced, as samples and reagents can be transferred to all 96 wells at once. This enables rapid and efficient, high throughput PCR clean-up.

  • Table of contents

    QIAquick 96 PCR Purification Kit is suitable for purifying up to 10 µg of material for downstream applications, such as sequencing, cloning, labeling and microarrays. The kit facilitates the removal of impurities like primers, unincorporated nucleotides, buffers, salts, mineral oils, agarose and enzymes. The vacuum-driven process is much faster than centrifugation, and gives high, reproducible yields. It is important to avoid cross-contamination in nucleic acid purification, and QIAGEN's column design is optimized to limit carryover of contaminants. Although QIAquick 96 provides a high throughput solution, the elution, washing and binding steps are very laborious and time consuming if performed manually. With VIAFLO 96 handheld electronic pipette, the hands-on time is reduced, as samples and reagents can be transferred to all 96 wells at once. This enables rapid and efficient, high throughput PCR clean-up.

Key benefits

  • VIAFLO 96 and VIAFLO 384 allow simultaneous pipetting of up to 96 or 384 wells, respectively, maximize throughput of PCR purification by allowing transfer samples and reagents in a single step.
  • The z-heights can be predefined, choosing the optimal value to prevent accidental scratching of the well membrane for more consistent results.
  • Custom programming of the PCR product clean-up steps allows pipetting parameters, such as aspiration or dispensing speeds, to be predefined. Prompt messages guide the user through the entire pipetting protocol, which is especially useful when several pre-wetting steps are included.
  • The VIAFLO 96 or VIAFLO 384's hands-free automatic mode ensures that the PCR clean up protocols are performed in the same way each time, maximizing reproducibility.

Overview: How to purify PCR products with VIAFLO 96

Experimental set-up

This protocol describes how PCR products are purified using a VIAFLO 96 handheld electronic pipette with a two position stage and the QIAGEN QIAquick® 96 PCR Purification Kit. The following procedure is based on the kit manufacturer's protocol for purification of 96 samples (up to 10 µg PCR products).

A 96 channel pipetting head (50-1250 µl) is used together with 1250 µl SHORT, Low Retention, Sterile, Filter GripTips. Customized VIALINK programs are provided to perform the binding, washing and elution steps. Before starting, ethanol (96-100 %) should be added to the Buffer PE concentrate.

Overview of the purification steps:

  • Step 1: Binding
  • Step 2: Washing
  • Step 3: Elution

The initial set-up of the QIAvac 96 Vacuum Manifold consists of a waste tray on top of a QIAvac base, followed by a QIAquick  96 well plate (pink) mounted on a QIAvac 96 top plate, as shown in Figure 1
The QIAvac has to be attached to a vacuum source (house vacuum or vacuum pump) that generates negative pressure between 100 and 600 mbar.

Initial set-up of the vacuum manifold
Figure 1: Initial set-up of the vacuum manifold.

Downloads: App note and protocols for semi-automated PCR product purification

Here you can download the complete protocols used in this application and use them on your VIAFLO 96. The protocol can be customized with the VIALINK software.

 

Step-by-step procedure

1. Binding

Binding the DNA to the silica-gel membran

Load the 1250 µl SHORT, Low Retention, Sterile, Filter GripTips on the VIAFLO 96. Place a 150 ml Automation Friendly Reagent Reservoir in position A. The QIAvac 96 Vacuum Manifold should be placed on position B of the VIAFLO96 in landscape orientation. No plateholder is needed on position B where the manifold is placed.

Important: The vacuum manifold should be aligned before each run (Figure 2).

Begin by launching the custom VIALINK program 'Qiaquick_purification_M'. The pipette will prompt the user to place Buffer PM on position A, then air is aspirated. This ensures that every single drop of the liquid can be dispensed later. The VIAFLO96 will then guide the user through the two pre-wetting steps, starting with aspiration and dispensing 200 µl of Buffer PM. After a second aspiration, the pipette will display the prompt 'Move the head out of buffer', before dispensing the final 200 µl of Buffer PM. This is followed by a 20 second wait, giving the buffer residues time to flow down to the tip and be dispensed.

Alignment of the QIAvac 96 Vacuum Manifold
Figure 2: Alignment of the QIAvac 96 Vacuum Manifold.

After pre-wetting, the pipette aspirates 75 µl Buffer PM (three times the volume of the PCR product). The instrument then tells the user to remove the reservoir from position A, and replace it with the 96 well plate containing the 25 µl of PCR products. After dispensing, and four mixing steps, the resulting mixture is transferred to the QIAquick plate wells in two steps. It is then time to switch on the vacuum source, as indicated by the pipette.

Tips:

  • Pre-wetting the tips prior to pipetting prevents droplets and dripping when pipetting volatile liquids, such as isopropanol, which is one of the constituents in Buffer PM.
  • Low Retention GripTips (Figure 3) are used for these pipetting steps to avoid dripping.
Low retention versus standard tips
Figure 3: Low retention versus standard tips.

2. Washing

Two-step purification of the PCR product

Eject the used tips and load new 1250 µl SHORT, Low Retention, Sterile, Filter GripTips on the VIAFLO 96. Place a new 300 ml automation friendly reagent reservoir in position A. The VIAFLO 96 will then prompt the user to pour Buffer PE into the reservoir, followed by a pre-wetting step, which is necessary since the buffer contains ethanol. After pre-wetting, the pipette will aspirate 900 µl of Buffer PE, and dispense it into QIAquick plate wells. The instrument will then notify the user that is it time to turn on the vacuum pump. With the pump turned on, another dose of the buffer is dispensed into the wells, followed by a 10 minute wait to dry the membrane and remove all residual ethanol.

IMPORTANT: The final drying step is crucial to remove residual ethanol prior to elution. Residual ethanol in the elution buffer could inhibit downstream applications (e.g. PCR).

Tip:

  • After this step, the manufacturer suggests tapping the plate on a stack of absorbent paper to ensure that all residual buffer is removed.

3. Elution

Elution of DNA from the silica-gel membrane

When prompted, start by replacing the waste tray with the blue collection microtube rack provided, which contains 1.2 ml vessels (Figure 4 a)). Load new 1250 µl SHORT, Low Retention, Sterile, Filter GripTips, and place a new 150 ml automation friendly reagent reservoir in position A. The instrument will then prompt the user to place Buffer EB into the reservoir, aspirate 80 µl, and dispense it into the QIAquick plate wells. After a 1 minute incubation, the pipette tells the user to switch on the vacuum source for 5 minutes.

Tips:

  • The purified PCR product could also be eluted in a 96 well microplate. In this case, when replacing the waste tray, the 96 well microplate has to be placed on the empty blue collection tube rack (Figure 4 b)).
  • For increased DNA concentration, decrease the elution volume to 60 µl, as per QIAGEN's recommendations, in the VIALINK software.

Remarks

Vacuum manifold

Alignment of the vacuum manifold is very important in this process. Adding marks on the deck helps to reposition the manifold whenever needed. To check the position of the well plate on top of the vacuum manifold, attach the tips manually to the pipette. The pipette tips should be in the middle of the wells. If not, adjust the position of the vacuum manifold on the deck.

Automatic mode

The VIAFLO 96 can also operate in hands-free automatic mode, allowing the user to have more walk-away time and less interaction, which is highly beneficial when using the instrument in a laminar flow cabinet. The customized automatic VIALINK program can be found in the download section.

Conclusion

  • The VIAFLO 96 electronic handheld pipette allows fast and simple liquid transfers for high throughput PCR product purification.
  • Optimized pipette settings enable accurate sample and reagent transfer, without the tip touching and scratching the QIAquick membrane.
  • The VIAFLO 96 electronic handheld pipette's compact design takes up minimal space and fits on any lab bench.
  • The unique operating concept makes the VIAFLO 96 and VIAFLO 384 as easy to use as a conventional electronic pipette.
  • The QIAvac 96 manifold is easily placed on the instrument and allows the processing of other kits using 96 well silica-membrane or filter plates.
  • Another option for this application is the MINI 96, which is the most affordable 96 channel option on the market.

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  • Éva Mészáros, PhD

    Application Specialist

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Instruments and accessories

VIAFLO 96/ VIAFLO 384, Handheld Electronic Pipette GripTip, 1250 µl SHORT, Low Retention, Sterile, Filter (for automation systems) Plate holder, spring loaded, with slide function 96 Channel Pipetting Head, 1250 µl Automation Friendly Reagent Reservoirs, 150 ml Automation Friendly Reagent Reservoir, 300ml QIAGEN: QIAquick 96 PCR Purification Kit QIAGEN: QIAvac 96 Vacuum Manifold VACUUBRAND: Diaphragm pump MZ 2 NT

VIAFLO 96/ VIAFLO 384, Handheld Electronic Pipette

With this 24, 96 and 384 channel pipette, INTEGRA meets the need for an affordable, compact and easy to use pipette that provides increased productivity. The unique operating concept makes VIAFLO 96/384 as easy to use as any traditional handheld pipette.

Part No. 6001 / 6031

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The VIAFLO 96/384 is handheld, electronic pipette

GripTip, 1250 µl SHORT, Low Retention, Sterile, Filter (for automation systems)

GripTip pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

Part No. 6595

Learn more about GripTips pipette tips for Benchtop Pipetting Systems
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Plate holder, spring loaded, with slide function

For 24, 96 and 384 channel pipetting. Available for deck position A (6215) and B (6220), these plate holders feature a spring load mechanism to secure microplates in place. Both holders also feature the slide function to access 384 well plates with a 96 channel head.

Part No. 6215 / 6220

Overview plate holder options
Spring loaded plate holder A for 96 and 384 well plates, with slide function

96 Channel Pipetting Head, 1250 µl

Users can simply change the pipetting head of their VIAFLO 96 and VIAFLO 384 to guarantee optimal matching of the available volume range to the application performed. Changing a pipetting head of VIAFLO 96 and VIAFLO 384 is easy and takes less than one minute.

Part No. 6102

Find out more about VIAFLO 96/384 Pipetting Heads Specifications
VIAFLO 96/384 96 channel pipetting head

Automation Friendly Reagent Reservoirs, 150 ml

150 ml Automation Friendly Clear Advantage™ Reservoirs (Polystyrene) which are made of crystal clear 100 % virgin polystyrene and fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.

The SureFlo™ Array located on the flat bottom reservoir prevents pipette tip seal off. This allows users to rest the pipette tips directly on the bottom of the reservoir offering an incredibly low dead volume of less than 3 ml!

Find out more about Automation Friendly Reagent Reservoirs
Reservoir for automation with SureFlo™ anti-sealing array

Automation Friendly Reagent Reservoir, 300ml

INTEGRA offers two types of automation friendly reagent reservoirs:

  • 150 ml and 300 ml Clear Advantage™ reagent reservoirs which are made of both crystal clear virgin polystyrene and polypropylene. Both options fit into a reusable automation friendly base with bold, crisp, clearly visible graduation markings.
     
  • The 300 ml open well, 12 column and 8 row polypropylene reservoir blocks offer improved chemical compatibility.
Find out more about Automation Friendly Reagent Reservoirs
300 ml Reservoir filled

QIAGEN: QIAquick 96 PCR Purification Kit

QIAquick 96 PCR Purification Kits provide 96-well plates, buffers, and collection tubes for high-throughput silica-membrane-based purification of PCR products >100 bp in size. DNA up to 10 kb is purified using a simple and fast bind–wash–elute procedure and an elution volume of 60–80 μl (resulting in an eluate volume of 40–60 μl). The cleanup procedure can be fully automated on the BioRobot workstations using the QIAquick 96 PCR BioRobot Kit.

Part No. 28181

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QIAquick 96 PCR Purification Kit

QIAGEN: QIAvac 96 Vacuum Manifold

QIAGEN QIAvac vacuum systems enable fast and efficient vacuum processing of up to 24 spin columns using the QIAvac 24 Plus, of 96-well plates using the QIAvac 96 and up to 6 HiSpeed Mega or Giga Columns using the QIAvac HiSpeed LS Vacuum.

Part No. 19504

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QIAGEN: QIAvac 96 Vacuum Manifold

VACUUBRAND: Diaphragm pump MZ 2 NT

Diaphragm pumps are an excellent solution for continuous, oil-free evacuation and pumping of gases. All parts in contact with pumped media are made of aluminum and selected plastics and allow a wide range of applications for non corrosive gases.

Part No. MZ 2 NT

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VACUUBRAND: Diaphragm pump MZ 2 NT

Customer’s voice

The device is user friendly with simple software which can easily be taught to anyone. We've replaced lot of manual pipetting steps with VIAFLO 96/384 and became more efficient and our TAs happier. We've also scaled up the tests we are doing and established new assays and applications that we haven't had before.

NATASA STOJANOVIC, Fraunhofer ITEM-R