Chromatin IP DNA Purification Kit(version A3)
Catalog No. 58002

Copyright 2014 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
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The manufacturer of this documentation is Active Motif, Inc.
© 2014 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respectivecompanies.
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Chromatin IP DNA Purification Kit Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocols
A. Purification of DNA from Chromatin IP Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Appendix
Section A.
Section B.
Section C.
Section D.

Supplementary Chromatin IP Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
DNA Recovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Frequently Asked Questions (FAQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful, well established technique for studying
interactions between chromatin-associated proteins and specific regions of the genome. The useof ChIP in combination with genome-wide analysis techniques can yield a tremendous amountof information regarding the distribution of transcription factors and histone modifications. But,
these techniques require DNA that has been purified away from the components and contaminants present in an eluted ChIP sample. Active Motif’s Chromatin IP DNA Purification Kit enables
you to quickly clean up your ChIP DNA samples and get them ready for analysis, without the needfor messy, labor intensive and time consuming phenol/chloroform extraction.
The Chromatin IP DNA Purification Kit is designed to be used to purify DNA following chromatin
immunoprecipitation. It contains all necessary reagents to purify DNA from 50 ChIP reactions andcan be used with material derived from any of the Active Motif line of ChIP-IT® Kits, including ourChIP-IT® Protein G Magnetic Beads (Catalog No. 53014).

product

formatcatalog no.

Chromatin IP DNA Purification Kit50 rxns

58002

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Kit Components and Storage
Please store each component at the temperature indicated in the table below.
Reagents

Quantity

Storage / Stability

DNA Purification Binding Buffer

50 mlRoom temperature

DNA Purification Wash Buffer*

50 ml

Room temperature

DNA Purification Elution Buffer

5 mlRoom temperature

3M Sodium Acetate**

500 µlRoom temperature

DNA purification columns

50

Room temperature

DNA column collection tubes

50

Room temperature

Columns and buffers (with the exception of the 3M Sodium Acetate) can be stored at 4ºC, but allcomponents must be warmed to room temperature before use. Some of the salts present in thebuffers may precipitate out of solution during storage and will need to be resuspended prior to use.
* DNA Purification Wash Buffer must be reconstituted to a final concentration of 80% ethanol
prior to use.
** Do not store the 3M Sodium Acetate at 4ºC.

Additional materials required• Ethanol, 100%
• Microcentrifuge• 1.5 or 2 ml microcentrifuge tubes

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Chromatin IP DNA Purification Kit Experimental DesignPLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Prior to using the Chromatin IP DNA Purification Kit for the first time, the DNA Purification Wash
Buffer must be reconstituted to a final concentration of 80% ethanol using 100% ethanol suppliedby the user. This only needs to be performed once, prior to first using the kit. After reconstitution,
store the buffer at room temperature. See step A1 in on page 4.
The Chromatin IP DNA Purification Kit is designed for use with a microcentrifuge for sample
processing but can also be used with a vacuum manifold. Please consult the manufacturer’sinstructions for adaptation of the kit for vacuum manifold use.
Compatibility: Once your ChIP experiments are complete, the DNA purification procedure can bestarted immediately. The entire procedure should take no more than five to ten minutes, depending upon the number of samples to purify. The Chromatin IP DNA Purification Kit is compatible
with samples from all Active Motif ChIP-IT Kits, or from any standard chromatin IP kit or procedure, using either mechanical or enzymatic shearing of chromatin, agarose or paramagneticbeads. Before purification, DNA samples should be eluted from the beads, cross-link reversed,
and, if the ChIP procedure calls for it, Proteinase K and/or RNase digested.
Binding of DNA to Column: One critical aspect of the procedure is the pH of the sample when
it is added to the column. Binding of DNA to the silica matrix of the DNA purification column will
only occur at or below pH 7.5. A pH indicator dye is present in the DNA Purification Binding Buffer
so that you can easily monitor that your samples are at the correct pH. To ensure successful
purification of your ChIP DNA, sodium acetate (pH 5.4) is supplied in case the pH of your sample
needs to be adjusted lower.
Washing: DNA bound to the column is extensively washed after binding.
Elution: After washing, the DNA is eluted using the included DNA Purification Elution Buffer.
Elution can be carried out using highly pure water (pH 7) if desired.
Purity: Use of the Chromatin IP DNA Purification Kit results in recovery of purified DNA of highquality suitable for a number of downstream analysis techniques, including PCR (endpoint orquantitative), Southern blotting, microarray, standard or Next-Gen sequencing or cloning.
Recovery: DNA fragments below 50 base pairs in length are not recovered efficiently.
Yield: Recovery of DNA will be 85-100%. Depending upon the number of cell equivalents ofchromatin used in each ChIP reaction, this typically will be from 100 ng to 1 µg. DNA can be
successfully recovered from ChIP experiments starting with as few as 10,000 cells.

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3Protocols
A. Purification of DNA from Chromatin IP Samples
1.

ADD ETHANOL BEFORE THE FIRST USE ONLY! The final concentration of ethanol in theDNA Purification Wash Buffer must be 80%. Add 40 ml of fresh 100% ethanol to the DNA
Purification Wash Buffer bottle. Invert repeatedly. The buffer can be stored at room temperature after the addition of ethanol.

2.

Your eluted ChIP samples should be in an Eppendorf tube. To each sample, add 5 volumes
DNA Purification Binding Buffer for every volume of DNA sample. For example, if your ChIP
sample is 100 µl, add 500 µl Binding Buffer.

3.

Add 5 µl 3M Sodium Acetate and mix. Check that the color of the DNA sample / DNA Purification Binding Buffer mixture is bright yellow, not light orange or violet. If the color is lightorange or violet, this indicates that its pH is too high. Add more 3M Sodium Acetate
5 µl at a time, mixing after addition, until the color is bright yellow (see Figure 1). As theelution buffers used in many ChIP protocols (including those in the ChIP-IT Kits) have abasic pH, this step is crucial to the successful binding and purification of your ChIP DNA
sample.
Figure 1: Solution color as a function of pH.
The DNA Purification Binding Buffer has a pH indicator dye,so that the pH of the solution can be easily determined. Only
apply the sample to the column when the solution is brightyellow (left), indicating a pH under 7.5. DNA will not bind tothe column if the pH is higher than 7.5.
Note: A full-color PDF of this manual can be downloaded
from the Active Motif website, which will enable you to see
this figure in color.

4.

For each sample, place a DNA purification column in the collection tube and add eachsample to its own column. Close the cap on each column, place them with the collection
tubes in a microcentrifuge and spin them at 14,000 rpm for 1 minute. Alternatively, a compatible vacuum manifold may be used for sample processing.

5.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

6.

Add 750 µl of Wash Buffer to the column and cap the column.

7.

Spin at 14,000 rpm for 1 minute in a microcentrifuge.

8.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

9.

With the column cap open, spin at 14,000 rpm for 2 minutes in a microcentrifuge to remove
any residual Wash Buffer from the column.

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410. Transfer the column to a clean Eppendorf tube.
11.

Add 50 µl of DNA Purification Elution Buffer (or purified water with a pH equal to or greaterthan 7) to the center of the column matrix and wait for 1 minute.
Note:

The amount of Elution Buffer used can be reduced to less than 50 µl in order
to attain a more concentrated DNA sample, if required. Reducing the elution
volume will result in an overall reduction in the recovery efficiency of thepurification. For more information, please see Appendix, Section B, Figure 5.

12. Spin at 14,000 rpm for 1 minute in a microcentrifuge.
13. Discard column. The DNA eluted into the Eppendorf tube is purified and ready to use in yourapplication.

Appendix
Section A. Supplementary Chromatin IP DataThe Chromatin IP DNA Purification Kit is designed for purification of DNA after performing a ChIP
experiment. Below are examples of quantitative PCR (qPCR) data from DNA purified using theChromatin IP DNA Purification Kit after ChIP.

Figure 2: Quantitative PCR performed on ChIP DNA purified with the Chromatin IP DNA Purification Kit.
Quantitative PCR was performed using DNA purified with the Chromatin IP DNA Purification Kit after ChIP using theindicated antibodies (ChIP Abs). Chromatin IP experiments were performed using the ChIP-IT® Express Kit (Catalog No.
53008) and Ready-to-ChIP HeLa Chromatin (Catalog No. 53015, 7.5 x 105 cell equivalents per ChIP). Quantitative PCR wascarried out using primers specific for the indicated gene (Locus) and normalized data represent fold enrichment over ChIP
experiments carried out with control IgG.

www.activemotif.com5Section B. DNA Recovery
The Chromatin IP DNA Purification Kit enables recovery of DNA fragments as small as 50 basepairs (Figure 3). As nucleosomal DNA is on the order of 150 base pairs, all of your chromatin IP
DNA should be recovered efficiently.
Figure 3: DNA recovery as a function of fragment size.
The Chromatin IP DNA Purification Kit enables efficient recovery of DNA fragments
as small as 50 base pairs. DNA molecular weight markers (10 base pair ladder) werepurified with the Chromatin IP DNA Purification Kit and run on a 5% agarose gel andstained with ethidium bromide to visualize the recovered DNA fragments.
Lane 1: DNA not purified.
Lane 2: DNA after purification.
As the figure illustrates, DNA larger than 50 base pairs is represented equally in thepurified and non-purified DNA samples. DNA fragments smaller than 50 base pairs(bracket) are not efficiently purified, and thus are not represented in the purified DNA
sample (Lane 2, bracket).

Figure 4: Concentration of DNA recovered as a function of elution volume.
Human genomic DNA was purified using the Chromatin IP DNA Purification Kit and eluted using the indicated volume of
Elution Buffer and the concentration (ng/µl) of the recovered DNA was determined.

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Figure 5: Yield of DNA recovered as a function of elution volume.
Human genomic DNA was purified using the Chromatin IP DNA Purification Kit and eluted using the indicated volume of
Elution Buffer and the quantity (ng) of the recovered DNA was determined.

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7Section C. Troubleshooting Guide
Problem/question

Recommendation

No amplification by PCRof ChIP DNA

If you are experiencing problems with downstream analysis of ChIP DNA, it ispossible that the purification process was compromised and your DNA samples
were not recovered. This could be due to one of several factors.
1. The pH of the Binding Buffer was too high.
Refer to Figure 1 on page 4 to make sure that the pH of your sample isbelow 7.5 to ensure proper binding of ChIP DNA to the column. Your
sample should be bright yellow (not orange, peach or purple). Add 3M
Sodium Acetate in aliquots of 5 µl until the pH reaches the proper point
and the sample is bright yellow. (A full-color PDF of this manual can be
downloaded from the Active Motif website, which will enable you to see
what is meant by “bright yellow”.)
2. No ethanol in the DNA Purification Wash Buffer.
Make sure that ethanol has been added to the DNA Purification Wash Buffer prior to use. See step A1 on page 4.
3. Residual DNA Purification Wash Buffer in the column prior to elution.
Make certain to spin the column one extra time after washing to remove
any residual Wash Buffer, as it will interfere with elution of the DNA fromthe column. Open the cap of the column to enable proper removal ofresidual Wash Buffer.
4. Salt in the Elution Buffer.
DNA will not be eluted from the silica matrix of the DNA purification column if the buffer used for elution contains high salt. Use the included DNAPurification Elution Buffer or purified water (pH 7) for elution.

Section D. Frequently Asked Questions (FAQ)
Can the Chromatin IP DNA Purification Kit be...
used after 5-hydroxymethylcytidine MeDIP? Yes.
used after 5-methycytidine MeDIP? Yes.
Note:

The MeDIP procedure using the 5-Methylcytidine antibody (Catalog No. 39649)
requires the DNA to be made single stranded. Single-stranded DNA does notbind as efficiently to the silica matrix of the column so recovery may not be100%, but our kit has been shown to work as well as the leading competitor’skit.

used with other Active Motif kits, such as the hMeDIP Kit and the MethylCollector™ family? Yes.
used to purify DNA samples from other manufacturer’s ChIP kits? Yes.
used to purify DNA samples from non-kit based ChIP protocols? Yes.
used with a vacuum manifold? Yes.
used to purify RNA? Not recommended. The buffers are not guaranteed to be RNase-free.
used to purify PCR products? Yes.
used to clean up restriction enzyme reactions? Yes.
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8Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
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Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

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