AMPure XP Bead-Based Reagent Protocol for PCR Purification

The protocol describes procedures of purification (cleanup) of PCR products in 96 and 384 well format using the AMPure XP reagent powered by our SPRI technology. This reagent utilizes an optimized buffer to selectively bind DNA fragments 100 bp and larger to paramagnetic beads. Excess primers, nucleotides, salts, and enzymes can be removed using a simple washing procedure.

NOTE: Although AMPure XP size selection protocols developed by other organizations do exist, we cannot guarantee performance or support this application. We suggest using the SPRIselect reagent, as it is developed specifically for size selection purposes. Want to learn more about the difference between the AMPure XP and SPRIselect reagents? Visit this page.

PCR Reaction Volumes
96 Well Format (μL)		 Product Number
A63880		 Product Number
A63881		 Product Number
A63882
10 278 rxns 3332 rxns 25000 rxns
20 139 rxns 1666 rxns 12500 rxns
50 56 rxns 667 rxns 5000 rxns
100 28 rxns 334 rxns 2500 rxns
 
PCR Reaction Volumes
384 Well Format (μL)		 Product Number
A63880		 Product Number
A63881		 Product Number
A63882
5 556 rxns 6667 rxns 50000 rxns
7 397 rxns 4762 rxns 35714 rxns
10 278 rxns 3333 rxns 20000 rxns
14 198 rxns 2381 rxns 17857 rxns

Consumables and Hardware

NOTE: To learn more about automation of the AMPure XP PCR Purification system on the Biomek i5 Multichannel 96 Genomics Workstation, read this application note.

Reagents

  • Fresh 70% ethanol
  • Reagent Grade Water (Either water, TRIS-Acetate (10 mM pH 8.0), or TE Buffer (10 mM Tris-Acetate pH 8.0, 1 mM EDTA) for DNA elution

96 Well Format Procedure

  1. Determine whether or not a plate transfer is necessary.

    If the PCR reaction volume multiplied by 2.8 exceeds the volume of the PCR plate, a transfer to a 300 μL round bottom plate or a 1.2 mL deep-well plate is required.

  2. Shake the AMPure XP bottle to resuspend any magnetic particles that may have settled. Then add the AMPure XP reagent according to the sample reaction volume shown in the table:

    Sample Reaction Volume (μL) AMPure XP Volume (μL)
    10 18
    20 36
    50 90
    100 180

    The volume of the AMPure XP reagent for a given reaction can be derived from the following equation:

    (Volume of AMPure XP per reaction) = 1.8 x (Reaction Volume)

    Learn here what the bead ratio is and why it matters.

  3. This step binds DNA fragments 100 bp and larger to the magnetic beads. Pipette mixing is preferable to vortexing as it tends to be more reproducible. The color of the mixture should appear homogenous after mixing:

    —Mix reagent and sample thoroughly by pipette mixing 10 times. Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery.

  4. Place the reaction plate onto the SPRIPlate 96R Ring Super Magnet Plate for 2 minutes to separate beads from the solution.

    IMPORTANT: Wait for the solution to clear before proceeding to the next step.

  5. This step must be performed while the reaction plate is situated on the SPRIPlate 96R Ring Super Magnet Plate:

    —Aspirate the cleared solution from the reaction plate and discard. Leave 5 μL of supernatant behind, otherwise beads are drawn out with the supernatant.

    IMPORTANT: Do not disturb the ring of separated magnetic beads.

  6. IMPORTANT: Perform this step with the reaction plate situated on the SPRIPlate 96R Ring Super Magnet Plate. Do not disturb the separated magnetic beads. Also, be sure to remove all of the ethanol from the bottom of the well.

    Dispense 200 μL of 70% ethanol to each well of the reaction plate and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard.

    NOTE: If the total volume of sample plus reagent exceeds 200 μL, then use a wash volume of at least the volume of sample plus reagent.

    Repeat for a total of two washes.

    The beads are not drawn out easily when in alcohol, so it is not necessary to leave any supernatant behind.

    NOTE: A dry time is optional to ensure all traces of ethanol are removed. For fragments 10 kb and larger, do not over dry the bead ring (bead ring appears cracked if over dried) as this will significantly decrease elution efficiency.

  7. Remove the reaction plate from the magnet plate, and then add 40 μL of elution buffer to each well of the reaction plate and pipette mix 10 times. Incubate for 2 minutes.

    The liquid level will be high enough to contact the magnetic beads at a 40 μL elution volume. A greater volume of elution buffer can be used, but using less than 40 μL will require extra mixing (to ensure the liquid comes into contact with the beads), and may not be sufficient to elute the entire PCR product.

  8. Place the reaction plate onto the SPRIPlate 96R Ring Super Magnet Plate for 1 minute to separate beads from the solution.

  9. Transfer the eluate to a new plate.

    NOTE: Bead carryover into the final plate is usually not a cause for concern. The samples can be stored in the freezer with beads and the beads are inert in downstream enzymatic reactions. If bead carryover must be limited for any reason, 2 μL – 5 μL of eluate can be left behind in the original plate. In addition, a second transfer away from the beads is optional. To do so, place the final plate containing beads and eluate onto the magnet for 1 minute to separate the beads. Transfer the eluate into another clean plate.

  1. Shake the AMPure XP bottle to resuspend any magnetic particles that may have settled. Then add the AMPure XP reagent according to the sample reaction volume shown in the table below.

    NOTE: Due to the total volume of sample plus reagent, it is not possible to purify reactions larger than 14 μL within the well of a 384 well PCR plate as shown in the following equation:

    (14 μL reaction + 25 μL AMPure XP = 39 μL)

    Sample Reaction Volume (μL) AMPure XP Volume (μL)
    5 9
    7 12.6
    10 18
    14 25

    The volume of the AMPure XP reagent for a given reaction can be derived from the following equation:

    (Volume of AMPure XP per reaction) = 1.8 x (Reaction Volume)

  2. This step binds DNA fragments 100 bp and larger to the magnetic beads. Pipette mixing is preferable to vortexing as it tends to be more reproducible. The color of the mixture should appear homogenous after mixing:

    —Mix reagent and sample thoroughly by pipette mixing 10 times. Let the mixed samples incubate for 5 minutes at room temperature for maximum recovery.

  3. Place the reaction plate onto the SPRIPlate 384 Post Magnet Plate for 2 minutes to separate beads from the solution.

    IMPORTANT: Wait for the solution to clear before proceeding to the next step.

  4. This step must be performed while the reaction plate is situated on the SPRIPlate 384 Post Magnet Plate:

    —Aspirate the cleared supernatant from the reaction plate and discard. Leave 5 μL of supernatant behind, otherwise beads are drawn out with the supernatant.

    IMPORTANT: Do not touch the magnetic beads, which have formed a spot on the side of the well.

  5. IMPORTANT: Perform this step with the reaction plate situated on the SPRIPlate 384 Post Magnet Plate. Do not disturb the separated magnetic beads. Also, be sure to remove all of the ethanol from the bottom of the well.

    Dispense 30 μL of 70% ethanol wash solution to each well of the reaction plate and incubate for 30 seconds at room temperature. Aspirate the ethanol out and discard.

    NOTE: If the total volume of sample plus reagent exceeds 200 μL, then use a wash volume of at least the volume of sample plus reagent.

    Repeat for a total of two washes.

    The beads are not drawn out easily when in alcohol, so it is not necessary to leave any supernatant behind.

    NOTE: A dry time is optional to ensure all traces of ethanol are removed. For fragments 10 kb and larger, do not over dry the bead pellets (bead pellets appear cracked if over dried) as this will significantly decrease elution efficiency.

  6. Remove the reaction plate from the magnet plate, and then add 30 μL of elution buffer to each well and pipette mix 10 times. Incubate for 2 minutes.

    A 30 μL elution volume will ensure the liquid level will be high enough to contact the magnetic beads. A greater volume of elution buffer can be used, but using less than 15 μL requires extra mixing (to ensure the liquid comes into contact with the beads) and may not fully elute the entire product.

  7. Place the reaction plate onto the SPRIPlate 384 Post Magnet Plate for 1 minute to separate beads from the solution.

  8. Transfer the eluate to a new plate.

    NOTE: Bead carryover into the final plate is usually not a cause for concern. The samples can be stored in the freezer with beads and the beads are inert in downstream enzymatic reactions. If bead carryover must be limited for any reason, 2 μL – 5 μL of eluate can be left behind in the original plate. In addition, a second transfer away from the beads is optional. To do so, place the final plate containing beads and eluate onto the magnet for 1 minute to separate the beads. Transfer the eluate into another clean plate.

For further assistance, please refer to the AMPure XP Instructions for Use or contact our expert by completing the form.

Products and demonstrated applications are not intended or validated for use in diagnostic procedures.

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