Diffinity RapidTip® D1947L
Product Information Sheet

for PCR purification
Compatible with Rainin LTS® pipettes

Product Description

The Diffinity RapidTip contains everything needed for PCR purification. The functional tip contains a proprietary adsorption technology which has a differential affinity for PCR reaction components. The impurities are removed from the solution as it enters the pipette tip and, after mixing for just one minute, dispensing the solution yields purified, high quality DNA ready to use for downstream applications.

Product Use

Diffinity RapidTip is designed for purification of PCR solutions prior to dideoxy (BigDye®) reaction for Sanger Sequencing. This RapidTip is compatible with Rainin LTS pipettes. Diffinity RapidTip effectively removes up to 90% of dNTPs, primers, and primer dimers while providing up to 90% recovery of pure DNA fragments ranging in length from 100 bp to 10 Kb.The RapidTip is not compatible with detergents, mineral oil, or ready-toload style PCR master mixes that contain a density increasing compound.

Reagents Supplied

Material Catalog Number Quantity
Diffinity RapidTip for PCR
Purification, Compatible with
Rainin LTS pipettes
D1947L 8 RXN
48 RXN
96 RXN

Equipment and Reagents Required But Not Provided

  • PCR Sample
  • Tubes to store purified DNA
  • Pipettor- manual or programmable
  • Standard pipette tips for liquid transfer (if needed)

Precautions and Disclaimer

The Diffinity RapidTip is for R&D use only, not for drug, household, or other uses. Consult the Safety Data Sheet (SDS) for information regarding hazards and safe handling practices.

Guidelines for Pipetting with the RapidTip

Careful pipetting is important to achieve effective purification with the RapidTip. The following pipetting guidelines will help ensure optimal results.

  1. Pre-wet the particles
    On the first aspirate step, aspirate approximately the first half of the sample and pause 5 seconds to ensure  complete wetting of the particles before mixing. Then aspirate the rest of the sample and dispense.

  2. Aspirate slowly
    For effective mixing, it is important to aspirate slowly with successive cycle to ensure that particles remain in contact with the sample.

    If using a manual pipettor, aspirate slowly for the first several cycles until the particles are completely dispersed into the PCR sample.

    If using an automated pipettor, set to aspirate on low speed (2 of 10 or equivalent), dispense on middle speed (5 of 10 or equivalent) and place into manual mix mode.

  3. Maintain tip-to-sample contact
    During aspiration, maintain continuous contact between the end of the pipette tip and your sample to avoid aspirating air bubbles in the sample.

  4. Maintain tip-to-well clearance
    Maintain clearance between the end of the pipette tip and the bottom of the sample tube or well to avoid plugging the tip during sample mixing.

  5. Recovering from particle detachment
    If particles become detached from the sample, gently “flick” the pipettor downward to bring them back into  contact with the sample and proceed with mixing.

Storage/Stability

Store the tips at room temperature.

Procedure

Prepare Samples:

Diffinity RapidTip is optimized for 25 µL reaction volumes but can effectively purify samples from 20- 30 µL.

  • For PCR volumes > 30 µL, aliquot 25 µL into each tube for purification.
  • For PCR volumes < 20 µL, dilute to 25 µL using 1x PCR buffer. This works best with highly concentrated DNA samples (>50 ng/uL) as your sample concentration will be reduced.

Prepare tips:

Diffinity RapidTip contains proprietary particles that purify a PCR reaction; these particles can adhere to the pipette tip walls during shipping. For optimal results, sharply tap the box 2-3 times on a flat surface so that particles are at the bottom of the tips (near the retainer).

Please note that it is normal to see fine dust-like particles on the side of the tip. After tapping the box, expect to see about 2 mm of white particles above the retainer at the small end of the pipette tip.

Purify Samples:

  1. Program pipettor to aspirate 50 µL
  2. Place Diffinity RapidTip on pipettor.
    Note: A multi-channel pipettor can be used to mix more than 1 sample at a time for even higher productivity.
  3. Place pipette tip into 25 µL PCR solution.
  4. Pre-wet the particles on first aspirate (see Pipetting Guidelines)
  5. Mix for 60 seconds (approximately 15 aspirate / dispense cycles).
    Note: Pipetting will be slower than normal. Wait for liquid to completely fill the tip to begin the next mix. It is not necessary to drive liquid completely out of the tip on every dispense. The particles should mix completely with the solution and make it appear cloudy.
  6. Dispense all solution into a clean tube when mixing is complete. Use your pipettor’s blowout mode for maximum liquid recovery.

The purified PCR amplicon is now ready for downstream Sanger sequencing. If you wish to confirm purification, run unpurified and purified sample in adjacent lanes on a gel to confirm the amplicons band. To estimate percent recovery, analyses of the samples pre- and post-purification are necessary. For this process, we recommend either a PicoGreen® 2 type assay or visualization on an agarose gel.

Troubleshooting Guide

Problem Solution
Low DNA Recovery Run some of the untreated and RapidTip purified PCR on a gel to check amplicon band intensity.
Note: Purify at lease 20 mL of the PCR reaction to optimize DNA recovery.
Slow or difficult aspiration Check that the pipettor volume is 50 μL
Check that the tips are firmly attached to the pipettor.
Note: Diffinity RapidTip is incompatible with detergents, mineral oil, and ready load PCR mixes that contain density increasing compounds.
Fluid remains in the RapidTip It is normal for a small amount of liquid (~5 μL) to remain inside the Diffinity RapidTip.
Note: Over-dispense or blow out all fluid on the last dispense cycle.
Failure to remove impurities Verify that particles are at the bottom of the tip prior to sample treatment; the pipettor is operating at the proper (slow) speed settings; and that particles are effectively mixing with the sample during pipetting.
Note: Remove trapped air bubbles by tapping the tip or mix for additional
aspirate/dispense cycles.

Material

     


BigDye is a registered trademark of Applera Corp.
PicoGreen is a registered trademark of Molecular Probes, Inc.
RapidTip is a registered trademark of Diffinity Genomics, Inc.
LTS is a registered trademark of Rainin Instrument, LLC.

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