iPure kit v2
Magnetic DNA Purification kit
for epigenetic applications
Cat. No. C03010014 (x24)Cat. No. C03010015 (x100)

USER GUIDE
V508_2023

Please read this manual carefully
before starting your experimentContentsIntroduction

4

Kit Method Overview

5

Workflow description

6

Kit Content

7Required Materials Not Provided

8

Protocol9

IPure after ChIP
STEP 1A - Chromatin Reverse Crosslinking and Elution

10

STEP 2A - DNA Binding

12

IPure after MeDIP
STEP 1B - DNA Elution

13

STEP 2B - DNA Binding

15

IPure after CUT&Tag
STEP 1C - pA-Tn5 Inactivation and DNA Release

16

STEP 2C - DNA Binding

17

IPure after ChIP, MeDIP and CUT&Tag
STEP 3 - Washes

18

STEP 4 - DNA Elution

20Results

21

Related Products

22

Introduction
Diagenode’s IPure kit is the only DNA purification kit using magnetic
beads, that is specifically optimized for extracting DNA from ChIP,
MeDIP and CUT&Tag (Chromatin Immunoprecipitation, Methylated DNA
Immunoprecipitation and Cleavage Under Targets & Tagmentation).
It’s a simple and straightforward protocol that delivers pure DNA readyfor any downstream application (e.g. next generation sequencing). Thisapproach guarantees a minimal loss of DNA and reaches significantly
higher yields than a column purification (see results pag). Comparing tophenol-chloroform extraction, the IPure technology has the advantage ofbeing nontoxic and much easier to be carried out on multiple samples.
The use of the magnetic beads allows for a clear separation of DNA andincreases therefore the reproducibility of your DNA purification.
The kit IPure can be used with the Diagenode's DiaMag02 magnetic rack:

DiaMag02 - magnetic rackCat. No. B04000001• Holds 16x standard 0.2 ml tubes• Working volume: 25-200 µl

4

IPURE KIT V2 MANUALTOTAL TIME

DNA elution

4 hours
30 min15 minSTEP 4.

Washes1 min

10 min

4 hours orovernight

STEP 3.

DNA binding

STEP 2A.

Chromatin reverse
cross-linking
and elution

STEP 1A.

IPure after ChIP

DNA elution

STEP 4.

WashesSTEP 3.

DNA binding

STEP 2B.

DNA elution

STEP 1B.

IPure after MeDIP

1 hour15 min

1 min

10 min30 minDNA elution

STEP 4.

WashesSTEP 3.

DNA binding

STEP 2C.

pA-Tn5 inactivation
and DNA release

STEP 1C.

1 hour15 min

1 min10 min

30 minIPure after CUT&Tag

Kit Method Overview

5Workflow description
IPure after ChIP
Step 1: Chromatin is decrosslinked and eluted from beads (magnetic oragarose) which are discarded. Magnetic beads for purification are added.
Step 2: Magnetic beads acquire positive charge to bind the negatively
charged phosphate backbone of DNA. DNA-bead complex is separatedusing a magnet.
Step 3: Proteins and remaining buffer are washed away.
Step 4: DNA is eluted from magnetic beads, which are discarded. Purified
DNA is ready for any downstream application (NGS, qPCR, amplification,microarray).
IPure after MeDIP
Step 1: DNA is eluted from beads (magnetic or agarose) which arediscarded. Magnetic beads for purification are added.
Step 2: Magnetic beads acquire positive charge to bind the negatively
charged phosphate backbone of DNA. DNA-bead complex is separatedusing a magnet.
Step 3: Remaining buffer are washed away.
Step 4: DNA is eluted from magnetic beads, which are discarded. Purified
DNA is ready for any downstream application (NGS, qPCR, amplification,microarray).
IPure after CUT&Tag
Step 1: pA-Tn5 is inactivated and DNA released from the cells. Magnetic
beads for purification are added.
Step 2: Magnetic beads acquire positive charge to bind the negatively
charged phosphate backbone of DNA. DNA-bead complex is separatedusing a magnet.
Step 3: Proteins and remaining buffer are washed away.
Step 4: DNA is eluted from magnetic beads, which are discarded. Purified
DNA is ready for any downstream application (NGS, qPCR, amplification,microarray).
6IPURE KIT V2 MANUAL

Kit Content
The kit content is sufficient to perform 24 (C03010014) or 100 reactions(C03010015).
IPure Kit
Description

Quantity (x24)

Quantity (x100)

Storage200 µl tube strips (8 tubes/strip)+ cap strips

4 pc

15 pc

Room temperature

Buffer A

2.8 ml10 ml

4°C

Buffer B

115 µl460 µl4°C

Wash buffer 1 w/o isopropanol

1.5 ml

9 ml

4°C

Wash buffer 2 w/o isopropanol

1.5 ml9 ml

4°C

Buffer C

1.2 ml

9 ml

4°C

IPure Beads v3

240 µl

1 ml

4°C

Carrier*48 µl

200 µl-20°C*This product is shipped at 4°C. Store it at -20°C upon arrival. This is an optimizedbuffer (NOT CARRIER DNA).

7

Required Materials Not Provided• DiaMag02 - magnetic rack (B04000001)• Microcentrifuge for 0.2 ml tubes or for 1.5 ml tubes with correspondingadaptor.
• 100% isopropanol• Sodium Acetate• 70% ethanol• 100% ethanol• DNase-free water• For CUT&Tag:• 0.5M EDTA
• 10% SDS
• Thermolabile proteinase K (eg New England Biolabs P8111S)

8IPURE KIT V2 MANUALPROTOCO L
IPure after ChIP
STEP 1A - Chromatin Reverse Crosslinking and Elution

10STEP 2A - DNA Binding

12IPure after MeDIP
STEP 1B - DNA Elution

13

STEP 2B - DNA Binding

15

IPure after CUT&Tag
STEP 1C - pA-Tn5 Inactivation and DNA Release

16

STEP 2C - DNA Binding

17

IPure after ChIP, MeDIP and CUT&Tag
STEP 3 - Washes

18

STEP 4 - DNA Elution

20

IPure after ChIP

STEP 1A
Chromatin Reverse Crosslinking
and Elution
After the last wash of immunoprecipitated material, discard the last tracesof wash buffer and use the pellet of beads (8-tube strip) for STEP 1.1.
1.1

Prepare the Elution Buffer by mixing Buffer A and B as follows:
Elution Buffer1 rxn

Buffer A

115.4 µl

Buffer B

4.6 µlTotal Volume120 µl• Place Buffer A at 25°C during 30 minutes before use.
• 100 µl of Elution Buffer are needed per IPure reaction (20 µlexcess).
• 1 IPure reaction corresponds to the purification of 1 ChIP or 1
input sample.
NOTE: Make sure when working with Buffer A, that there are no crystals leftin solution. Otherwise heat up gently and mix until complete disappearence ofsuch crystals.

1.2

10Add 100 µl of Elution Buffer to the bead pellets (tube strip).

IPURE KIT V2 MANUALThaw your input sample (1.5 ml tube), and perform a pulse spin. Add
90 µl of Elution Buffer and 10 µl of input sample to a new 200 µl
tube (8 tube-strip).
NOTE: Input sample corresponds to 10% of the IP sample.

1.4

Incubate samples and input for 4 hours (or overnight) at 65°C on athermomixer, with continuous shaking.

1.5

Spin the 8-tube strip and place it into the DiaMag02 magnetic rack.
After 1 minute, transfer the supernatants to a new labelled 8-tubestrip. Keep the samples on ice.

DETAILED PROTOCOL

1.311

IPure after ChIP

STEP 2ADNA Binding
2.1

Add 2 µl of carrier to each IP and input sample. Vortex briefly andperform a short spin.

2.2

Add 100 µl of 100% isopropanol to each IP and input sample. Vortex
briefly and perform a short spin.
NOTE: Following the addition of isopropanol the solution may become cloudy.
This is not detrimental to your experiment and will not influence the quality or
quantity of your purified DNA.

2.3

Resuspend the provided Magnetic beads and transfer 10 µl to each
IP and input sample.
• Keep Magnetic beads in liquid suspension during storage at4°C and at all handling steps, as drying will result in reducedperformance.
• The final volume is now 212 µl per IPure reaction.

2.4

12Incubate IP and input samples for 10 minutes at room temperature
on a rotating wheel (40 rpm).

IPURE KIT V2 MANUALSTEP 1B
DNA Elution

DETAILED PROTOCOL

IPure after MeDIP

After the last wash of immunoprecipitated material, discard the last tracesof wash buffer and use the pellet of beads (8-tube strip) for STEP 1.1.
1.1Prepare the Elution Buffer by mixing Buffer A and B as follows:Elution Buffer

1 rxn

Buffer A

115.4 µl

Buffer B

4.6 µlTotal Volume120 µl• Place Buffer A at 25°C during 30 minutes before use.
• 100 µl of Elution Buffer are needed per IPure reaction (20 µlexcess).
• 1 IPure reaction corresponds to the purification of 1 MeDIP or 1
input sample.
NOTE: Make sure when working with Buffer A, that there are no crystals leftin solution. Otherwise heat up gently and mix until complete disappearence ofsuch crystals.

1.2

Add 50 µl of Elution Buffer to the bead pellets (tube strip).

1.3

Thaw your input sample (1.5 ml tube), and perform a pulse spin. Add
92.5 µl of Elution Buffer and 7.5 µl of input sample to a new 200 µl
tube (8-tube strip).
NOTE: Input sample corresponds to 10% of the IP sample.
13

1.4

Incubate samples and input DNA for 15 minutes at room temperature
on a rotating wheel (40 rpm).

1.5

Spin the 8-tube strip and place it into the DiaMag02 magnetic rack.
After 1 minute, transfer the supernatants to a new labelled 8-tubestrip.

1.6

Repeat the incubation of the bead pellets for 15 minutes at room
temperature on a rotating wheel (40 rpm) in 50 µl Elution Buffer.
• For input DNA samples: 1 elution in 100 µl.
• For MeDIP samples: 2 elutions in 50 µl (total volume 100 µl).

1.7

Spin the 8-tube strip. Place the 8-tube strips into the DiaMag02
containing the 50 µl IP samples, wait 1 minute and transfer thesupernatants to the new labelled 8-tube strip to pool with thecorresponding IP samples.
• Elutions of IP and input samples are now completed in 100 µl andare in the same 8-tube strip.

14IPURE KIT V2 MANUALSTEP 2BDNA Binding
2.5

Add 2 µl of carrier to each IP and input sample. Vortex briefly andperform a short spin.

2.6

Add 100 µl of 100% isopropanol to each IP and input sample. Vortex
briefly and perform a short spin.

DETAILED PROTOCOL

IPure after MeDIP

NOTE: Following the addition of isopropanol the solution may become cloudy.
This is not detrimental to your experiment and will not influence the quality or
quantity of your purified DNA.

2.7

Resuspend the provided Magnetic beads and transfer 10 µl to each
IP and input sample.
• Keep Magnetic beads in liquid suspension during storage at
4°C and at all handling steps, as drying will result in reduced
performance.
• The final volume is now 212 µl per IPure reaction.

2.8

Incubate IP and input samples for 10 minutes at room temperature
on a rotating wheel (40 rpm).

15

IPure after CUT & Tag

STEP 1C
pA-Tn5 Inactivation and DNA Release
NOTE: the protocol below is compatible with CUT&TAG version described by
Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. CUT&TAG for efficient epigenomic
profiling of small samples and single cells. Nat Commun 10, 1930 (2019).
https://doi.org/10.1038/s41467-019-09982-5

16

1.1

After completion of the tagmentation reaction, add 10 µl of 0.5 M
EDTA, 3 µl of 10% SDS and 1 µl of thermolabile proteinase K directly
to each sample and mix by full-speed vortexing for 2 seconds. Do notremove the Tagmentation Buffer and Concavalin A beads.

1.2

Incubate at 37ºC for 15 minutes.

1.3

Heat samples at 55ºC for 10 minutes to inactivate thermolabile
proteinase K.

IPURE KIT V2 MANUALSTEP 2CDNA Binding

DETAILED PROTOCOL

IPure after CUT & Tag

NOTE: an equival volume of isopropanol should be added. If the tagmentation reaction
volume is different from 300 µl, adjust the required volume of isopropanol.

2.1

Add 300 µl of 100% isopropanol to each sample. Vortex briefly andperform a short spin. Do not remove Concavalin A beads. Following
the addition of isopropanol the solution may become cloudy. This isnot detrimental to your experiment and will not influence the quality
or quantity of your purified DNA.
NOTE: an equival volume of isopropanol should be added. If the tagmentation
reaction volume differs from 300 µl, adjust the required volume of isopropanol.

2.2

Re-suspend the provided Magnetic beads and transfer 10 μl to eachsample.
• Keep Magnetic beads in liquid suspension during storage at
4°C and at all handling steps, as drying will result in reduced
performance.
• The final volume is now 624 μl per IPure reaction.

2.3

Incubate samples for 10 minutes at room temperature on a rotating
wheel (40 rpm).

17

IPure after ChIP, MeDIP and CUT & Tag

STEP 3
Washes (ChIP, MeDIP and CUT&TAG)
3.1

Prepare the Wash Buffer 1 containing 50% isopropanol for 100
reactions:
Wash Buffer 124 rxns

100 rxns

Wash Buffer 1 w/o Isopropanol

1.5 ml9 ml

Isopropanol (100%)

1.5 ml9 ml

Total Volume

3 ml

18 ml

• Never leave the bottle open to avoid evaporation.
3.2

Briefly spin the tubes, place in the DiaMag02, wait 1 minute anddiscard the buffer. Keep the captured beads and add per tube, 100 µl
Wash Buffer 1. Close the tubes, invert the 8-tube strip to resuspend
the beads and incubate for 5 minutes at room temperature on arotating wheel (40 rpm).
• Do not disturb the captured beads attached to the tube wall.
• Always briefly spin the tubes to bring down liquid caught in the lid
prior to positioning into the Diagenode Magnetic Rack.

18IPURE KIT V2 MANUALPrepare the Wash Buffer 2 containing 50% isopropanol as follows:
Wash Buffer 224 rxns

100 rxns

Wash Buffer 2 w/o Isopropanol

1.5 ml9 ml

Isopropanol (100%)

1.5 ml9 ml

Total Volume

3 ml

18 ml

• Never leave the bottle open to avoid evaporation.
3.4

DETAILED PROTOCOL

3.3

Wash the IP and input samples with the Wash Buffer 2 as follows.
Briefly spin the tubes, place into the DiaMag02, wait 1 minute anddiscard the buffer. Keep the captured beads and add per tube, 100 µl
Wash Buffer 2. Close the tubes, invert the 8-tube strip to resuspend
the beads and incubate for 5 minutes at room temperature on arotating wheel (40 rpm).
• Do not disturb the captured beads attached to the tube wall.
• Always briefly spin the tubes to bring down liquid caught in the lid
prior to positioning in the DiaMag02.

19

IPure after ChIP, MeDIP and CUT & Tag

STEP 4DNA Elution (ChIP, MeDIP andCUT&TAG)
NOTE: This elution buffer (Buffer C) is suitable for direct qPCR analysis, wholegenome amplification, chip hybridization and next generation sequencing.

20

4.1

Briefly spin the tubes and place them into the DiaMag02, wait
1 minute and discard the buffer. Keep the captured beads and add
per tube, 25 µl of Buffer C (alternatively, a higher volume can beused for the elution if necessary). Close the tubes, invert the 8-tube
strip to resuspend the beads and incubate for 15 minutes at room
temperature on a rotating wheel (40 rpm). Resuspend the pelleted
beads using the pipet and make sure that you drop them on thebottom of the tube.

4.2

Spin the 8-tube strips and place it into the DiaMag02, wait 1 minuteand transfer the supernatants into a new labelled 1.5 ml tube. Keep
the bead pellets on ice.

4.3

Place the DNA on ice and proceed to any desired downstream
applications, or store it at -20°C or -80°C until further use.

IPURE KIT V2 MANUAL

Results
DNA recovery after purification of ChIP samples using IPure technology
1817.05 ng

16DNA recovery (ng)1412.95 ng

12108641.96 ng

200.2 ng
1,000 cells

10,000 cells

100,000 cells

300,000 cells

ChIP assays were perfomed using different amounts of U2OS cells, theLowCell# ChIP kit and the H3K9me3 antibody (Cat. No. C15410056; 2 μg/IP). The purified DNA was eluted in 50 μl of water and quantified with aNanodrop.

21

Related Products
Description

Cat. No.

FormatDiaMag02 - magnetic rack

B04000001

1 pc

200 µl tube strips (8 tubes/strip) + cap stripsC30020002

120 pciDeal ChIP-seq kit for Histones x10

C01010050

10 rxns

iDeal ChIP-seq kit for Transcription Factors x10

C01010054

10 rxns

MagMeDIP kit x10

C02010020

10 rxns

MagMeDIP kit x48

C02010021

48 rxns

pA-Tn5 loaded

C01070001

15 µl/30 µl

pA-Tn5 unloaded

C01070002

10 µl/30 µl

Revision history

22Version

Date of modification

V5 08_2023

August 2023

Correction of small error page 17, step 2.2.

V5 02_2023

February 2023

- Replacement of the IPure beads v2 by IPure beads v3- Removal of obsolete reference (DiaMag1.5)

IPURE KIT V2 MANUAL

Description of modifications

FOR RESEARCH USE ONLY.
Not intended for any animal or human therapeutic or diagnostic use.
© 2023 Diagenode SA. All rights reserved. No part of this publication may be reproduced, transmitted,transcribed,stored in retrieval systems, or translated into any language or computer language, in any
form or by any means:electronic, mechanical, magnetic, optical, chemical, manual, or otherwise,
without prior written permission from Diagenode SA (hereinafter, “Diagenode”). The informationin this guide is subject to change without notice. Diagenode and/or its affiliates reserve the rightto change products and services at any time to incorporate the latest technological developments.
Although this guide has been prepared with every precaution to ensure accuracy, Diagenode and/or its affiliates assume no liability for any errors or omissions, nor for any damages resulting fromthe application or use of this information. Diagenode welcomes customer input on corrections andsuggestions for improvement.
NOTICE TO PURCHASERLIMITED LICENSE
The information provided herein is owned by Diagenode and/or its affiliates. Subject to the terms andconditions that govern your use of such products and information, Diagenode and/or its affiliates
grant you a nonexclusive, nontransferable, non-sublicensable license to use such products andinformation only in accordance with the manuals and written instructions provided by Diagenodeand/or its affiliates. You understand and agree that except as expressly set forth in the terms andconditions governing your use of such products, that no right or license to any patent or otherintellectual property owned or licensable by Diagenode and/or its affiliates is conveyed or implied
by providing these products. In particular, no right or license is conveyed or implied to use theseproducts in combination with any product not provided or licensed to you by Diagenode and/or its
affiliates for such use. Limited Use Label License: Research Use Only The purchase of this productconveys to the purchaser the limited, non-transferable right to use the product only to perform
internal research for the sole benefit of the purchaser. No right to resell this product or any of
its components is conveyed expressly, by implication, or by estoppel. This product is for internal
research purposes only and is not for use in commercial applications of any kind, including, without
limitation, quality control and commercial services such as reporting the results of purchaser’s
activities for a fee orother form of consideration. For information on obtaining additional rights,
please contact info@diagenode.com.
TRADEMARKSThe trademarks mentioned herein are the property of Diagenode or their respective owners.
Bioanalyzer is a trademark of Agilent Technologies, Inc. Agencourt and AMPure® are registeredtrademarks of Beckman Coulter, Inc. Illumina® is a registered trademark of Illumina®Inc; Qubit is a
registered trademark of Life Technologies Corporation.

www.diagenode.com