Diffinity Genomics RapidTip2 User Handbook Manual

USER HANDBOOK

Version B (March 2012)

For PCR Purication with

Polymerase Removal

User Notice:

For optimal purication results, sharply tap
the RapidTip2 package 2–3 times on a at
surface so that the particles drop to the
bottom of the tips.

Table Of Contents

Kit Contents 3
Storage 3
Product Use Limitations 4
Product Warranty and Satisfaction Guarantee 4
Quality Control 5
Technical Assistance 5
Safety Information (Disposal) 5
Product Specifications 6
Introduction 7
Diffinity Technology Principle 8
Equipment to be Supplied by User 9
Guidelines — Pipetting with the RapidTip2 10
Pipetting Examples 11
RapidTip2 PCR Purification Protocols 12
Quantifying DNA Recovery 13
Troubleshooting Guide 14
Ordering Information 15

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Kit Contents

Diffinity RapidTip2s can be used with universal or Rainin LTS
style pipettes. All kits come with a user handbook.

Cat Number Product Description Contents

RR050-008
RR050-008L*

RR050-048
RR050-048L*

RR050-096
RR050-096L*

* L — denotes Rainin LTS format pipette tips.

Diffinity RapidTip2 for
PCR Purification with
Polymerase Removal

Diffinity RapidTip2 for
PCR Purification with
Polymerase Removal

Diffinity RapidTip2 for
PCR Purification with
Polymerase Removal

Storage

1 Pack of 8
RapidTip2s

1 Box of 48
RapidTip2s

1 Box of 96
RapidTip2s

Diffinity RapidTip2s should be stored in low humidity at room
temperature (15–25 degrees Celsius). Under these conditions,
Diffinity RapidTip2s can be stored and used for up to 12
months without reduction in performance or quality. An
expiration date is on the label of the 48 and 96 tip boxes.

Difnity RapidTip2 Page 3

Product Use Limitations

Diffinity RapidTip2s are intended for research use only. Diffinity
RapidTip2 is designed for purification of 50 μl PCR solutions
prior to dideoxy (BigDye) reaction for Sanger Sequencing,
restriction digestion, or T-A cloning. RapidTip2 is compatible
with most universal and Rainin LTS pipettors. RapidTip2 is not
compatible with detergents, mineral oil, or ready-to-load style
PCR master mixes that contain a density-increasing compound.

Product Warranty and Satisfaction Guarantee

Diffinity Genomics will guarantee the performance of Diffinity
RapidTip2s for the uses and performance as described in
our product literature. If the product fails to perform to your
satisfaction for any reason other than misuse, we will replace
the product free of charge or refund the purchase price —
whichever you choose. Diffinity Genomics maintains the right
to update or modify the product to enhance its performance. A
copy of our terms and conditions can be found on our web site.

If you have questions about the product or its performance,
please contact Diffinity Genomics customer service at
1-877-362-1812.

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Quality Control

Each lot of Diffinity RapidTip2s is tested against predetermined
specifications to ensure consistent quality.

Technical Assistance

Call 1-877-362-1812 between 8am and 4pm Eastern Time
Monday through Friday or visit www.diffinitygenomics.com for
help with common questions.

Safety Information (Disposal)

Diffinity RapidTip2s may be disposed in regular lab trash. For
more information, please consult the material safety data sheet
(MSDS) available on request by calling Customer Service at
1-877-362-1812.

Difnity RapidTip2 Page 5

Product Specications

Capacity: PCR Sample Size 50 ±5 microliters

dsDNA Recovery up to 90%

(100bp to 10Kb)

Impurity Removal (dNTP,
primers, primer-dimers)

DNA Polymerase removal
(Taq, Hot-Start, High­Fidelity)

up to 90%
(less than 40–50 bp)

up to 1:1000 dilution with
no measurable amplification
activity

Diffinity RapidTip2 tips are designed to remove dNTPs,
primers, primer-dimers, and DNA polymerase at typical PCR
concentrations.

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Introduction

Diffinity Genomics has developed an innovative new
technology that removes undesirable impurities from PCR
reactions leaving you with nothing but purified DNA!

This revolutionary technology effectively removes dNTPs,
primers, primer-dimers, and DNA polymerase while providing
up to 90% recovery of pure DNA fragments ranging in length
from 100bp to 10Kb. It dramatically reduces the amount of
time to purify and prepare PCR reactions for use in downstream
applications, such as Sanger sequencing, restriction digestion,
and/or T-A cloning.

The RapidTip family of tips for PCR purification require just
one-tip, one-step, and one-minute for PCR purification!

Difnity RapidTip2 Page 7

Difnity™ Technology Principle

The Diffinity RapidTip2 contains everything you need for PCR
purification. The tip is filled with our 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. Dispensing the solution yields purified,
high quality DNA ready to use for downstream applications.
You won’t need additional equipment, reagents, buffers, or
enzymes — only a pipettor!

Difnity RapidTip2 puries PCR
solutions and removes DNA
polymerase comparably to
popular spin column methods.

+ -C R

Standard

The positive control (+) con­tains polymerase and all PCR
reagents but did not undergo
purication and therefore has
a 600bp band. The control
purication (C) and RapidTip2
purication (R) do not have
bands showing that polymerase
was removed during purica­tion.

Hot Start

Proofreading

---RT2---

Difnity RapidTip2 effectively
removes dNTPs (not visible on
gel), primers (20bp), and primer­dimers (50bp). The PCR product
was divided and subjected to PCR
purication with either competitor’s
products (C) or RapidTip2 (RT2: 1,
2, 3). RapidTip2 has high yield: the
PCR amplicon (2Kb) shows no loss
of band intensity after purication.

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U C C 1 2 3

2Kb--

50bp-­20bp--

Diffinity RapidTip2
purification
improves sequence
quality. Sequence
chromatograms
show improved
peak quality and
lower background in
Diffinity RapidTip2
purified samples
as compared to
unpurified samples.

unpuried

RapidTip2

Equipment to be Supplied by User

• PCR Sample (50 μl ±5 μl)

• Tubes to store purified DNA

• Pipettor — manual or programmable

• Standard pipette tips for liquid transfer
(if necessary)

Difnity RapidTip2 Page 9

Guidelines — Pipetting with the RapidTip2

Careful pipetting is important to achieve effective purification
with the RapidTip2. 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. Mix effectively

For effective mixing, it is important that the particles

remain in contact with the sample during each aspirate and
dispense cycle.

Aspirate slowly for the first several cycles until the particles

are completely dispersed into the PCR sample.

Avoid long delays between successive aspirate and dispense

cycles that allow the particles to settle out of solution for
more than 1–2 seconds.

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.

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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 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.

Pipetting Examples

A. Correct mixing:

Sample and particles
mixing well.

Please note that typical PCR samples are colorless — for visualization,
yellow dye was used.

To correct B and to improve performance, gently “flick” the
pipettor downward to bring all particles and the sample back
together, then proceed with mixing.

Difnity RapidTip2 Page 11

B. Incorrect mixing:

Particles separated by
air bubble.

RapidTip2 PCR Purication Protocols

Prepare Samples:

Diffinity RapidTip2 is optimized for 50 μl reaction volumes but
can effectively purify samples from 45–55 μl.

• For PCR volumes >55 μl, please aliquot 50 μl into a new
tube for purification.

• For PCR volumes <45 μl, please dilute to 50 μl. This works
best with highly concentrated DNA samples (>50ng/μl)
as your sample concentration will be reduced.

Prepare Tips:

Diffinity RapidTip2 for PCR Purification 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 nozzle).

Please note that it is normal to see fine dust-like particles on the side of the
tip. After tapping the box, you should expect to see about 3mm of white
particles above the retainer at the small end of the pipette tip.

Purify Samples:

1. Program pipettor to aspirate 50 or 60 μl???.

2. Place Diffinity RapidTip2 on pipettor — single or
multichannel.

Please note that you can use a multi-channel pipettor to mix more than
1 sample at a time for even higher productivity.

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3. Place pipette tip into 50 μl PCR solution.

4. Pre-wet the particles on first aspirate (see page 10).

Aspirate about half the sample and then pause for 5 seconds to ensure
complete wetting of the particles before mixing. Aspirate the rest of the
sample and then dispense.

5. Set timer and mix for 60 seconds (approximately 15
aspirate/dispense cycles).

Please note that 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
while inside the tip.

6. Dispense all solution into a clean tube when mixing
is complete. Use your pipettor’s blowout mode for
maximum liquid recovery.

Your purified PCR Amplicon is now ready for your downstream
application.

Quantifying DNA Recovery

If you wish to confirm purification, run unpurified and purified
sample in adjacent lanes on a gel to confirm the amplicon
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

agarose gel.

Difnity RapidTip2 Page 13

Troubleshooting Guide

Problem Things to try:

Low DNA
recovery

Slow or
difficult
aspiration

Fluid
remains
in the
RapidTip2

Run some of the untreated and RapidTip2 purified
PCR on a gel to check your amplicon band
intensity.

Note: Purify at least 45 μl of your PCR reaction to
optimize DNA recovery.

Check that pipettor volume is 50 or 60 μl??.
Check that tips are firmly attached to the pipettor.

Note: Diffinity RapidTip2 is incompatible with
detergents, mineral oil, and ready-to-load PCR mixes
that contain density increasing compounds.

It’s normal for a small amount of liquid (~10μl) to
remain inside the Diffinity RapidTip2.

Note: Overdispense or blowout 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 gently “flicking”
the pipettor downward for additional aspirate/dispense
cycles.

Visit www.diffinitygenomics.com/FAQs.asp for additional
information.

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Ordering Information

You can place an order by:

1. Contacting one of our distributors worldwide. For a
complete list, visit our website.

2. Call Diffinity Genomics Customer Service and place the
order by phone: [phone]

3. Visit our website and place an order online via the online
Diffinity store: www.diffinitygenomics.com/order.asp

For more information, please visit our website:

www.diffinitygenomics.com

Difnity RapidTip2 Page 15

Difnity Genomics RapidTip2 User Handbook
Version B (March 2012)

Printed in the United States

Difnity Genomics, Inc.
150 Lucius Gordon Drive, West Henrietta, NY 14586

© 2011 Difnity Genomics, Inc. All rights reserved. Difnity
Genomics, Difnity and RapidTip are trademarks of Difnity
Genomics, Inc. All other names may be trademarks or
registered trademarks of their respective owners.