MethylCollector™ Ultra(version D3)

Catalog No. 55005

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
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2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2012 Active Motif, Inc.

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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 any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is
proprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2012 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 respective
companies.
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TABLE OF CONTENTS

Page

Overview  . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction  . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Traditional Methods to Study DNA Methylation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Fragmentation of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Example Fragmentation Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
MethylCollector™ Ultra Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Appendix
Section A. Downstream Applications  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section B. PCR Analysis
		
PCR Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
		
Endpoint PCR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
		Real Time PCR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
		Data Analysis and Use of Inputs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Section C. Use of Magnetic Beads and Included Bar Magnet  . . . . . . . . . . . . . . . . . . . . . . 23
Section D. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Section E. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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Overview
Active Motif’s MethylCollector™ Ultra Kit is an improved alternative for the enrichment of CpGmethylated DNA1 from limited amounts of cell or tissue samples. The method is based on the
Methylated CpG Island Recovery Assay (MIRA), which utilizes the high affinity of the MBD2b/MBD3L1 complex for methylated DNA2. MethylCollector Ultra is suitable for use in many downstream
applications, such as real time or endpoint PCR analysis of the methylation status of particularloci in normal and diseased samples, rapid screening of the methylation status of multiple loci,bisulfite conversion followed by cloning and sequencing, or amplification and labeling for microarray analysis3. It can also be used to detect changes in DNA methylation during normal cellulardifferentiation and aging.
In the MethylCollector Ultra method*, His-tagged recombinant MBD2b and its binding partner
MBD3L1 are combined together in order to increase the affinity of MBD2b for CpG-methylatedDNA. The kit works using DNA fragments that have been prepared by enzymatic digestion or sonication from cell or tissue samples. The MBD2b/MBD3L1 protein complex is added to the DNA fragments, and it specifically binds to CpG-methylated DNA. These protein-DNA complexes are thencaptured with nickel-coated magnetic beads and subsequent wash steps are performed to remove
fragments with little or no methylation. The methylated DNA is then eluted from the beads in thepresence of Proteinase K. Due to the high efficiency of MethylCollector Ultra and the enormous
amplification capability and specificity of PCR, analysis of the methylation status of a specificgenomic DNA locus can be performed on DNA isolated from less than 170 cells (~1 ng DNA).

product

formatcatalog no.

MethylCollector™ Ultra

30 rxns**

55005*Technology covered under U.S. Patent No. 7,425,415.
**MethylCollector™ Ultra provides sufficient reagents to perform 30 reactions withexcess reagents for 5 control reactions.

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Break open cells and shear
DNA by Sonication or
Enzymatic Shearing.

Incubate sheared DNA with
His-tagged MBD2b/MBD3L1
protein complexes.

Capture the methylated
protein-DNA complexes withnickel-coated magnetic beads.

methylatedDNA

The purified, methylated
DNA is readyfor PCR analysis.

Elute the bound, methylatedDNA, while simultaneously
degrading the protein complexes.

Wash to remove theunmethylated
DNA fragments.

Flow chart of the MethylCollector Ultra process.
In MethylCollector Ultra, genomic DNA of interest is sheared by either enzymatic digestion or sonication. The sheared
DNA is then incubated with a His-tagged recombinant MBD2b/MBD3L1 protein complex. The interaction of MBD2bwith its binding partner MBD3L1 increases the affinity of MBD2b for CpG-methylated DNA. These protein-DNA complexes are captured with nickel-coated magnetic beads and stringent washes are then performed to remove fragmentswith little or no methylation. The methylated DNA is then eluted from the beads and real time or endpoint PCR is
performed on the resulting supernatant using specific primers to amplify the locus of interest.

Introduction
Over the last decade, the study of DNA methylation and its role in epigenetic cell signaling has
grown rapidly4-7. Methylation of CpG dinucleotides, which occurs at the fifth position of thecytosine pyrimidine ring, is of particular interest.
Although CpG dinucleotides are generally methylated throughout the genome of normal somatic
cells, CpG islands (clusters of CpG dinucleotides in gene regulatory regions) are usually unmethylated8. Aberrant hypermethylation of CpG islands and subsequent transcriptional repression is oneof the earliest and most common somatic genome alterations in multiple human cancers9, 10. Somewhat paradoxically, a decrease in the total amount of cytosine methylation is observed in many
neoplastic tissues, but the genome context of this hypomethylation has not been identified11.
Aberrant methylation of CpG islands thus seems to be a tumor type-specific event10, 12 and current
efforts have concentrated on finding ways to exploit the diagnostic and therapeutic implicationsof these abnormalities13, 14.

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Methyl-CpG binding proteins appear to be central players in the process of DNA methylationdependent gene silencing15. This family of proteins takes its definition from the methyl-CpG binding domain (MBD), the minimum portion with specific affinity for a single, symmetrically methylated CpG pair. The MBD was characterized by deletion studies of MeCP216. After the recognitionof the MBD, four additional genes were found to contain this domain, namely MBD1, MBD2, MBD3
and MBD417. In general, all MBD proteins, except MBD4, have been reported to be associated withhistone deacetylase subunits as part of large multi-subunit complexes18, 19. A few studies support
the notion of selectivity in the association of a particular MBD with particular promoters20, 21, butother results indicate that the CpG distribution along the sequence may influence the interactionof each MBD protein with DNA22.
The MBD2b protein has been found to possess one of the highest affinities for methylated DNA
among MBD proteins and has the greatest capacity to differentiate between methylated and unmethylated DNA22. The combination of MBD2b with its binding partner MBD3L1, methyl-CpG-binding protein 3-like-1, generates a higher affinity for methylated DNA than MBD2b protein alone2, 23.
The specificity of the MBD2b/MBD3L1 complex is able to enrich for methylated DNA fragments
containing as few as 5 methylated CpGs, and is more efficient at methylated DNA enrichment
than antibody-based immunoprecipitation methods. In addition, MethylCollector Ultra can beused in parallel with the UnMethylCollector™ Kit (Catalog No. 55004) to provide a more thoroughanalysis of the methylation state at each locus of interest24.

Traditional Methods to Study DNA Methylation
To date, there are several methods used for methylation analysis:
1. Methylation-sensitive restriction enzyme analysis: Isoschizomers of bacterial restriction
endonucleases with different sensitivities for 5-methylcytosine can be used to determine
the methylation status of specific CpG dinucleotides25. Methylation-sensitive restriction
enzymes have several limitations, such as the fact that the methylation-sensitive restriction
merely informs on the methylation status of the cytosine residues which are recognized by
the restriction enzymes used, but do not provide information about other methylation sites.
2. Bisulfite conversion: Bisulfite conversion26 consists of the treatment of double-stranded
genomic DNA with sodium bisulfite, leading to deamination of unmethylated cytosines intouracil. PCR is then performed with primers that differentiate between methylated andunmethylated sequences. Bisulfite-based techniques can be cumbersome, involving timeand labor-intensive chemical treatments that damage DNA and limit throughput.
3. Methylated DNA Immunoprecipitation (MeDIP): In this assay, an antibody specific formethylated cytosines (anti-5-methylcytosine antibody) is used to immunoprecipitate methylated DNA from genomic DNA fragmented by enzymatic digestion or sonication27. The resulting enrichment is usually analyzed by PCR based methods; thus MeDIP can be combined with
DNA microarrays for genome-wide analysis of CpG methylation. However, this technique isrelatively time-consuming, requires a large amount of fragmented DNA starting material andonly works with denatured DNA.

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3Kit Performance and Benefits
The MethylCollector Ultra Kit is for research use only. Not for use in diagnostic procedures.
Range of detection: MethylCollector Ultra can be performed on 1 ng - 1 µg of fragmented
genomic DNA.
Sensitivity: Enriches methylated DNA fragments with as few as 5 methylated CpGs.
Nature of the MethylCollector Ultra Assay: MethylCollector Ultra is an improved technique forthe enrichment of methylated CpG islands. The methylation status of specific promoters contained within CpG islands can be analyzed using either endpoint or real time PCR analysis of thelocus of interest with customer designed PCR primers. Control human, male genomic DNA thatwas digested with Mse I is included in the kit along with PCR primers specific for both unmethylated and methylated promoters.
GAPDH Glyceraldehyde-3-phosphate dehydrogenase is important for metabolism. Because
this gene is often constitutively expressed, it is considered to be an actively transcribed housekeeping gene containing an unmethylated promoter in healthy tissues.
The region amplified by this primer pair is 69 base pairs and contains 7 CpGs.
Xist

X inactive specific transcript is a methylated promoter in human male genomic DNA.
The region amplified by this primer pair is 178 base pairs and contains 8 CpGs.

NBR2

Neighbor of BRCA1 gene 2 is located near the breast cancer gene BRCA1. Evidence
indicates that NBR2 and BRCA1 share a bi-directional promoter. This region of the
NBR2 gene is methylated in healthy tissues. The region amplified by this primer pair is
103 base pairs and contains 7 CpGs.

With high salt binding conditions, the Mse I digested control human, male genomic DNA provided
in the kit should have at least a 10-fold enrichment of methylated DNA bound and eluted fromthe protein complex as detected with the NBR2 PCR Primer Mix. This means that of the total
DNA recovered, there is ten times as much methylated DNA recovered in the eluted fraction ascompared to the unbound fraction for the same locus.
With low salt binding conditions, the Mse I digested control human, male genomic DNA provided
in the kit should have at least a 10-fold enrichment of methylated DNA bound and eluted fromthe protein complex as detected with the Xist PCR Primer Mix. This means that of the total DNA
recovered, there is ten times as much methylated DNA recovered in the eluted fraction as compared to the unbound fraction for the same locus.
There should be less than 5% of methylated DNA detected in the eluted fraction using the GAPDHPCR Primer Mix for either low or high salt binding conditions.

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A. NBR2 Amplification

B. Xist Amplification
Amplification Plot

Amplification Plot
Bound (low salt)

8

Bound (high salt)

10

Bound (low salt)

8

Unbound (low salt)

Bound (high salt)

Unbound (low salt)
Unbound (high salt)

Unbound (high salt)

5

∆Rxn

∆Rxn

7
6

6

4
3

4

2

2

1
0

0
2

6

10

14

18

Cycle

22

26

30

34

38

2

6

10

14

18

22

26

30

34

38

Cycle

C. GAPDH Amplification∆Rxn

Amplification Plot
11
10
9
8
7
6
5
4
3
2
1
0

Unbound (low salt)
Unbound (high salt)
Bound (low salt)
Bound (high salt)

2

6

10

14

18

22

Cycle

26 30

34

38

Figure 1: Real time PCR analysis of control PCR primer sets.
MethylCollector Ultra was performed using 100 ng of Mse I digested human, male genomic DNA under both low andhigh salt conditions. Eluted DNA was purified and analyzed using real time PCR for both methylated and unmethylated
promoters. A) Amplification plot using the provided NBR2 PCR primer mix with the unbound and eluted fractions. NBR2 ismethylated in the control DNA and shows early amplification of the eluted fractions. B) Amplification plot using the provided Xist PCR primer mix with the unbound and eluted fractions. Xist is methylated in the control DNA and shows early
amplification in the eluted fractions. C) Amplification plot using the provided GAPDH PCR primer mix with the unboundand eluted fractions. GAPDH is unmethylated in the control DNA and shows late amplification of the eluted fractions.

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140

120
NBR2

100
80
60
40

80
60
40
20

20
0

Xist
GAPDH

100

GAPDH

Enrichment (%)

Enrichment (%)

120

Unbound0

ElutedUnboundElutedLow Salt Binding Conditions

High Salt Binding Conditions

Figure 2: Fold enrichment of MethylCollector Ultra binding reactions.
MethylCollector Ultra was performed with 100 ng of Mse I digested human, male genomic DNA using both high and low
salt binding conditions. Unbound and eluted DNA was cleaned and analyzed in real time PCR. The amount of DNA recovered in the eluted fraction was divided by the amount of input DNA used in the binding reaction to produce a percent
enrichment. The methylated promoters NBR2 and Xist had greater than 10-fold enrichment of eluted DNA as compared tounbound DNA for the same locus, while the unmethylated GAPDH promoter was found almost exclusively in the unboundfraction.

A. NBR2 Amplification
High salt
Binding Buffer
200 bpLow salt
Binding Buffer

UnboundElutedElutedUnboundInput

100 bpB. Xist Amplification
Low salt
Binding Buffer
300 bpElutedUnbound

UnboundElutedInput

200 bpC. GAPDH Amplification
High salt
Binding Buffer
100 bpElutedElutedLow salt
Binding Buffer

Unbound
UnboundElutedElutedUnbound
Unbound

Input
Input

Figure 3: Specificity of the MBD2b/MBD3L1 protein complex for CpG methylated DNA.
MethylCollector Ultra was performed with 100 ng of Mse I digested human, male genomic DNA using both high and low
salt conditions. Eluted DNA was cleaned and analyzed in endpoint PCR for 36 cycles. The methylated NBR2 (A) and Xist (B)promoters are highly enriched in the eluted fraction while the unmethylated GAPDH (C) promoter is only detected in theunbound fraction, indicating the high specificity of MBD2b/MBD3L1. The Xist PCR Primer Mix has been optimized for use
with low salt binding conditions.
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Kit Components and Storage
Kit components arrive on dry ice. Upon receipt, we recommend storing each component at thetemperatures listed in the table below. The magnetic beads can be stored frozen upon arrival,
however, we recommend long-term storage at 4°C. Once the magnetic beads have been thawed,
do not re-freeze the beads.
Reagents

Quantity

Storage / Stability

His-MBD2b/MBD3L1 protein complex

350 µl

-20°C for 6 months

High Salt Binding Buffer

35 ml-20°C for 6 months

Low Salt Binding Buffer

35 ml-20°C for 6 months

Elution Buffer AM1

3.5 ml

-20°C for 6 months

Protease Inhibitor Cocktail

100 µl

-20°C for 6 months

Proteinase K

70 µl-20°C for 6 months

Proteinase K Stop Solution

70 µl-20°C for 6 months

Human, male genomic DNA (Mse I digested)(20 ng/µl)

250 µl

-20°C for 6 months

GAPDH PCR Primer Mix (2.5 pmol/µl)

400 µl

-20°C for 6 months

Xist PCR Primer Mix (2.5 pmol/µl)

400 µl

-20°C for 6 months

NBR2 PCR Primer Mix (2.5 pmol/µl)

400 µl

-20°C for 6 months

10X PCR Buffer

1.5 ml

-20°C for 6 months

10X PCR Loading Dye

1.5 ml

-20°C for 6 months

Magnetic Nickel Beads

350 µl

4°C for 6 months

Glycogen (20 mg/ml)

35 µl-20°C for 6 months

Bar Magnet

1

Room temperature

Mini Glue Dots

2 DotsRoom temperature

8-strip PCR tubes and caps

12 strips

Room temperature

Additional Materials Required• Fragmented DNA sample• Sample PCR primer sets
• Sterile DNase-free water• Filter pipette tips
• Microcentrifuge tubes and microcentrifuge• Magnetic stand. You can assemble a magnetic stand using the provided bar magnet and glue
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dots (see Appendix - Section C) or use commercially available stands• Rotisserie shaker• Phenol/chloroform• 5 M Ammonium acetate (see Troubleshooting Guide, Appendix - Section D, for details
regarding the use of 3 M sodium acetate, pH 5.2)
• 100% ethanol• 70% ethanol• dNTP mixture (5 mM each)• Taq polymerase (5 U/µl) (Example: New England Biolabs M0267L or GeneSpin STS-T1000)• SYBR Green mix for real time PCR analysis• PCR cycler• (Optional) 5M Betaine stock solution for use with endpoint PCR

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Protocols
NOTES BEFORE STARTING
Fragmentation of Genomic DNA
The provided His-MBD2b/MBD3L1 protein complex has an enhanced affinity to bind methylated
cytosines, particularly DNA fragments that contain five or more methylated cytosines. To enable clear interpretation of results, genomic DNA should be prepared such that DNA fragments
containing a CpG region of interest do not contain methylated cytosines outside of this region(see “Appendix D. Troubleshooting” for further discussion). DNA can be fragmented by restriction
digest or by mechanical means (e.g., sonication).
Restriction digestion is especially useful for analysis of individual CpG islands. The genomic DNA
is cut with a methylation-insensitive restriction enzyme(s) so that only CpGs of interest arecontained within a particular restriction fragment. This fragment should be long enough (75 bp
or longer) to allow for PCR analysis. Some useful methylation-insensitive restriction enzymes areshown in the below table. As might be expected, the enzymes whose recognition sites contain G
and C bases cut more frequently in CpG islands than enzymes whose sites are composed only of A
and T bases.

Recognition
Sequence

Number of fragments(per kb) in CpG islands

Number of fragments(per kb) in non-CpG islands

Mse I

TTAA

0.80

2.88

Bfa I

CTAG

1.56

1.55

Tas I

AATT

0.80

2.88

Csp6 I

GTAC

2.23

1.41

Mechanical fragmentation is ideal when a single DNA sample will be used for simultaneous
analysis of many CpG islands (e.g., when the isolated DNA will be analyzed by microarray methods)
or when a CpG region of interest is not flanked by suitable restriction sites. In general, the DNA
should be sheared to an average fragment size of less than 500 bp to minimize the number of CpG
islands on each fragment.
Example fragmentation protocols are provided for both restriction digest and mechanical fragmentation on page 10. We suggest using 4 µg of purified genomic DNA when performing restriction digestion and 20 µg of purified genomic DNA for sonication. MethylCollector Ultra reactions
can be performed on 1ng - 1 µg of fragmented genomic DNA.

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Example Fragmentation Protocols
Restriction digest
This protocol can be modified depending on the amount of isolated genomic DNA or the
restriction enzyme being used. We recommend preparing high-quality genomic DNA using acommercially available kit or a standard established protocol. The quality of the genomic DNA
can be assessed by agarose gel electrophoresis and DNA concentration can be determined by UV
spectrophotometry.
a)

Set up the following restriction digest (with Mse I as an example, New England Biolabs (NEB)):		Genomic DNA (400 ng/µl)
10X NEB Buffer 4		
100X BSA			Mse I (10 U/µl)		
dH2O			Total volume		

10 µl
10 µl
1 µl
1 µl
78 µl
100 µlNote 1:

The DNA volume may vary depending on its initial concentration.

Note 2:

MethylCollector Ultra has been used with as little as 1 ng of restriction-digested
genomic DNA. As a reference, a human cell contains about 6 picograms DNA;
1 ng of genomic DNA corresponds to 170 cells.

b)

Mix well by pipetting and incubate at 37°C for 2 hours to overnight.

c)Heat-inactivate Mse I by incubating the reaction mixture at 65°C for 20 minutes. If using analternative restriction enzyme that cannot be heat-inactivated, the DNA can be purified byphenol/chloroform extraction and precipitation, or through use of a DNA purification column. See “Appendix D. Troubleshooting” on page 25 for comments about heat-inactivation.
Note 1:

For greater accuracy, the digested DNA should be quantified.

Note 2:This digested DNA should be stored at -20°C until use.

Mechanical fragmentation (sonication)
Because Mse I or other restriction enzymes cannot always be used to fragment and isolate the
DNA sequences of interest, sonication of the genomic DNA is an alternative method. Sonication isrecommended for DNA fragmentation in preparation for Next-Gen sequencing analysis.
a)Pipette 20 µg genomic DNA into a 1.5 ml microcentrifuge tube and adjust final volume to
300 µl by addition of 10 mM Tris-HCl pH 8.5.

b)Using a tip probe sonicator, sonicate on ice with 15 pulses of 20 seconds (30% amplitude if
using Active Motif’s EpiShear™ Sonicator, Catalog Nos. 53051 & 53052), with a 20-second pause
on ice between each pulse. The sheared DNA can be visualized by ethidium staining after
electrophoresis on a 3% agarose gel. The majority of the DNA fragments should be between
100 and 350 bp in length.
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MethylCollector Ultra Protocol
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Step 1: Planning the experiment
1. Determine the number of reactions needed. For PCR or sequencing analysis, one reaction
per sample is usually sufficient. For recommendations on downstream applications, such as
whole genome amplification or Next-Gen sequencing, please refer to Appendix A.
2.

Two different binding buffers are included in the kit. For high salt conditions (high stringency
reactions) we recommend using High Salt Binding Buffer. For low salt conditions (lower
stringency reactions) we recommend using Low Salt Binding Buffer. It may be necessary to
perform experiments with both binding conditions to determine the optimal conditions forthe target of interest.

Step 2: Binding reaction
1. Thaw components from storage as needed for preparation. Keep all components on ice
when not in use.
2.

Prepare Complete Binding Buffer for the desired binding conditions.
For high salt binding conditions (high stringency): Prepare the appropriate amount ofComplete Binding Buffer according to the table below. Store on ice.
Reagent

One rxn

8 rxnsHigh Salt Binding Buffer

100 µl800 µlProtease Inhibitor Cocktail

0.5 µl

4 µl

Total Volume

100.5 µl

804 µlFor low salt binding conditions (lower stringency): Prepare the appropriate amount ofComplete Binding Buffer according to the table below. Store on ice.
Reagent

One rxn

8 rxnsLow Salt Binding Buffer

100 µl800 µlProtease Inhibitor Cocktail

0.5 µl

4 µl

Total Volume

100.5 µl

804 µlNote:
3.

The provided High and Low Binding Buffers are optimal for efficient capture of
DNA fragments that contain five or more methylated CpGs.

Dilute the fragmented DNA in water if necessary. The MethylCollector Ultra protocol can be
performed on a large range of sample DNA amounts (1 ng to 1 µg). We recommend 100 ng
because it gives robust results without requiring a large amount of DNA.

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4.

In this step, the Input DNAs that will be used in the final PCR step are prepared.
a.

If performing real time PCR: For the control genomic DNA provided in the kit, it isrecommended that several Input DNA concentrations be run in triplicate. Input DNA
should be tested at 0.01, 0.1, 1 and 10 ng/µl.

b.

If performing endpoint PCR: For the control genomic DNA provided in the kit, PCR
analysis is performed for 36 cycles on 25 ng of control DNA. The control DNA (provided
at 20 ng/µl) should be diluted to 5 ng/µl for use in Input PCR. This can be done bydiluting the DNA 1/4 in dH2O (e.g., 5 µl of 20 ng/µl DNA + 15 µl dH2O to make 5 ng/µlDNA). 5 µl of the 5 ng/µl DNA is used for Input PCR (see page 21).

Note:

Customer sample Input DNA can be treated similarly. If your locus-specific PCRprimers are efficient and PCR will be performed for 36 cycles, 25 ng of sample
DNA can be used for the Input PCRs. However, PCR primer efficiency varies andyou may want to try several amounts of Input DNA to be sure to obtain PCR
products from reactions still in the linear phase of amplification.

5.

Using the PCR tubes provided, fully resuspend magnetic beads by inverting, then aliquot a
10 µl slurry into each tube. If preparing more than 4 reactions, cap and re-invert the beads
after every 4 aliquots. (Note: When working with magnetic beads, pipette gently.)

6.

Binding Reaction: Add the remaining components in the order shown below to each PCR
tube. Pipet the His-MBD2b/MBD3L1 protein complex up and down several times to ensure
homogeneity before use. Prepare a positive control reaction using the provided Mse I
digested human, male genomic DNA.
Reagent

One rxn

Positive Control

Magnetic beads

10 µl

10 µl

Complete Binding Buffer

70 µl

75 µl

Fragmented genomic DNA

from 1 ng-1 µg(in a final volume of 10 µl)-

Control Human, male genomic DNA(Mse I digested)

-

5 µl

His-MBD2/MBD3L1 protein complex

10 µl

10 µl

Total Volume

100 µl100 µlNote:

8.

It is recommended to aliquot the provided His-MBD2b/MBD3L1 protein complex
into several small fractions for future use. Pipet the His-MBD2b/MBD3L1 protein
complex up and down several times to ensure homogeneity before making
aliquots. Store at -20°C.

Cap tubes and shake to mix thoroughly. Incubate on a rotisserie shaker for 1 hour at 4°C.

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12

Step 3: Wash beads
1. After the capture step is complete, spin the PCR tubes briefly and place tubes on a magnetic
stand to pellet beads to the side of the tube. If further analysis of the unbound fraction will
be performed, such as a comparison of the level of enrichment of methylated DNA, placesupernatant in a microcentrifuge tube and set aside at 4°C for DNA clean up in Step 5 of the
protocol. Otherwise, remove and discard the supernatant. To use the magnet provided in thekit, please see Appendix C.
2.

Wash beads four times with 200 µl Binding Buffer. Pipette 2-3 times gently to resuspend. Use
the same stringency Binding Buffer for the wash steps as was used for the binding reactions.
a.

Place tubes on magnetic stand and allow beads to pellet on the side of the tube.

b.

Carefully remove the supernatant and any residual bubbles.

c.

Add 200 µl Binding Buffer and resuspend the pellet completely by pipetting several
times. Ensure that the beads do not stick to the pipette tips. Depending on thestrength of the magnet being used, it may be necessary to remove the tubes from themagnet and place in a separate rack to fully resuspend the beads.

d.

Repeat steps a-c.

3.

In a microcentrifuge tube, prepare Complete Elution Buffer by adding 2 µl of Proteinase K to
98 µl of Elution Buffer AM1 for each reaction.

4.

After the final wash, place tubes on magnetic stand and allow beads to settle to the side.
Remove and discard supernatant without disturbing the beads.

Step 4: Recovery of methylated DNA fragments
1.

Resuspend washed beads with 100 µl Complete Elution Buffer by pipetting 2-3 times.

2.

Incubate samples at 50°C for 30 minutes. Every 10 minutes, invert the tubes to ensure thebeads are resuspended.

3.

During this incubation warm the Proteinase K Stop Solution at 37°C for 10 minutes.

4.

Quick spin the tubes. Place tubes in magnetic stand and allow beads to pellet onto tube
sides.

5.

Carefully transfer the supernatant to a new microcentrifuge tube.

6.

Add 2 µl of Proteinase K Stop Solution to each supernatant. Pipet 2-3 times to mix.

7.

Proceed to Step 5, DNA clean up, or else DNA can be stored at -20°C. If the DNA is stored at-20°C, it recommended to be reheated at 37°C for 10 minutes prior to use.

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13

Step 5: DNA clean up
Prior to use in downstream applications it is necessary to clean up the DNA. DNA clean up can be
performed using one of the methods listed below:• Purification columns – use PCR clean up kits such as Active Motif’s Chromatin IP DNA Purification Kit (Catalog No. 58002). Samples should be eluted in 50 µl volume, or other appropriate volume of water or buffer as needed for specific downstream applications.
For microarray analysis, use MinElute PCR Purification Kit (Qiagen part no. 28004) and elute in
10 µl volume to obtain more concentrated DNA.
For MIRA-Seq, use MinElute PCR Purification Kit (Qiagen part no. 28004), elute into 24 µl
volume of EB and use 20 µl of the purified DNA for the library preparation. Refer to Section
A of the Appendix for more information on processing samples for MIRA-Seq.
• Phenol/chloroform extraction followed by ethanol precipitation – follow the protocol
listed below
Phenol/Chloroform Extraction & Ethanol Precipitation
1. Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v/v) to the eluted
sample and, if desired, the unbound fraction from Step 2 No. 1.
2.

Vortex the tube at maximum speed for 15 seconds.

3.

Centrifuge the tube for 5 minutes at 12,000 x g at room temperature.

4.

Carefully transfer the top, aqueous phase to clean microcentrifuge tube without collecting
any of the lower organic phase or precipitate that may occur between the phases.

5.

To each sample add:
1 µl Glycogen (20 mg/ml) (included in the kit)
1 sample volume of 5 M ammonium acetate
2.5 sample volumes of 100% ethanol

6.

Mix well and incubate at -80°C for at least 2 hours.

7.

Centrifuge the tube for 20 minutes at 12,000 x g, 4°C.

8.

Carefully discard the supernatant without disturbing the pellet.

9.

Add 500 µl of cold 70% ethanol. Do not disturb the pellet.

10. Centrifuge the tube for 10 minutes at 12,000 x g, 4°C.
11.

Carefully discard the supernatant without disturbing the pellet.

12.

Air-dry the pellet for 5 minutes (do not completely dry the pellet).

13.

Resuspend the DNA pellet in 50 µl sterile DNase-free water., or use other appropriate volumes of buffer or water as needed for specific downstream applications.

14.

This eluted DNA can be used immediately in PCR or stored at -20°C. If the DNA is stored at-20°C, it will need to be reheated at 37°C for 10 minutes prior to use in downstream applications.
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14

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.

Bird, A.P. (1986) Nature, 321: 209-213.
Rauch, T. and Pfeifer, G. (2005) Lab. Investigation, 85: 1172-1180.
Rauch, T. et al. (2006) Cancer Res., 66: 7939-7947.
Henikoff, S. and Matzke, M.A. (1997) Trends Genet., 13: 293-295.
Wolffe, A.P. and Matzke, M.A. (1999) Science, 286: 481-486.
Reik, W. et al. (2001) Science, 293: 1089-1093.
Grewal, S.I. and Moazed, D. (2003) Science, 301: 798-802.
Antequera, F. and Bird, A. (1993) Proc. Natl Acad. Sci. USA, 90: 11995-11999.
Jones, P.A. and Baylin, S.B. (2002) Nature Rev. Genet., 3: 415-428.
Esteller, M. et al. (2001) Cancer Res., 61: 3225-3229.
Feinberg, A.P. and Vogelstein, B. (1983) Nature, 301: 89-92.
Costello, J.F. et al. (2000) Nat. Genet., 24: 132-138.
Esteller, M. (2005) J. Pathol., 205: 172-180.
Kalebic, T. (2003) Ann. N Y Acad. Sci., 983: 278-285.
Ballestar, E. and Wolffe, A.P. (2001) Eur. J. Biochem., 268: 1-6.
Nan, X. et al. (1993) Nucleic Acids Res., 21: 4886-4892.
Hendrich, B. and Bird, A. (1998) Mol. Cell. Biol., 18: 6538-6547.
Wade, P.A. et al. (1999) Nat. Genet., 23: 62-66.
Feng, Q. and Zhang, Y. (2001) Genes Dev., 15: 827-832.
Magdinier, F. and Wolffe, A.P. (2001) Proc. Natl Acad. Sci. USA, 98: 4990-4995.
El-Osta, A. et al. (2002) Mol. Cell. Biol., 22: 1844-1857.
Fraga, M.F. et al. (2003) Nucleic Acids Res., 31: 1765-1774.
Jiang, C.L. et al. (2004) J. Bio. Chem., 279: 52456-52464.
Acevedo, L.G. et al. (2011) Epigenomics, 3(1): 93-101.
Dalh, C. and Guldberg, P. (2003) Biogerontology, 4: 233-250.
Frommer, M. et al. (1992) Proc. Natl Acad. Sci. USA, 89: 1827-1831.
Weber, M. et al. (2005) Nat. Genet., 37: 853-832.
Notes:The polymerase chain reaction (PCR) process for amplifying nucleic acid is covered by U.S. Patent Nos. 4,683,195 and 4,683,202 assigned to Hoffmann-La Roche.
Patents pending in other countries.
Use of methylation-specific PCR (MSP) is protected by U.S. Patent Nos. 5,786,146,
6,017,704, 6,200,756 & 6,265,171 and International patent WO97/46705. No licenseunder these patents to use the MSP process is conveyed to the purchaser bypurchasing this product.
MethylCollector™ Ultra is covered under U.S. Patent No. 7,425,415.

www.activemotif.com

15

Appendix
Section A. Downstream Applications
The heterodimeric MBD2b/MBD3L1 protein used in the MethylCollector Ultra Kit can be used to
selectively bind and enrich for methyl CpG dinucleotides that are often found in the promoter/regulatory regions of genes. The binding affinity of the MBD2b/MBD3L1 protein increases with thedensity of methyl CpG dinucleotides. The amount of DNA recovered from the MethylCollector
Ultra Kit will depend on the global methylation status of the sample DNA. Usually only a small
percentage of the starting material will be recovered, yet the recovered sample is specific for
CpG-methylated DNA.
PCR Analysis
By amplifying a specific target site using PCR, it is possible to determine if there is an increase
in methylation at that single site. Alternatively, the same site can be compared across different
sample types, but it will be necessary to run MethylCollector Ultra on the same amount of eachDNA sample and generate a separate standard curve for each sample.
Next-Gen Sequencing Analysis (MIRA-Seq)
To analyze whole-genome methylation profiles, researcher may chose to perform Next-Gen
sequencing following the enrichment of methylated DNA with MethylCollector Ultra. This process
has been validated when DNA enriched from MethylCollector Ultra is end adapted for use in
sequencing. For instructions on how to prepare eluted DNA for Next-Gen sequencing, please referto the Illumina ChIP-Seq DNA Sample Prep Kit protocol that can be viewed and downloaded at
http://support.illumina.com/sequencing/sequencing_kits/chip-seq_dna_sample_prep_kit/best_practices.ilmn.
Please refer to page 14 for recommended DNA clean-up and volume specifications for MIRA-Seq.
Microarray Analysis
Researchers who are interested in the global changes to methylation pattern due to an experimental treatment rather than specific target analysis may prefer to analyze the enriched methylated DNA by microarray, also known as MIRA-chip. For an overview of this technology, please visit
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html.
MethylCollector Ultra can be used prior to microarray analysis. The final elution of the MBD2b/
MBD3L1 pulled-down DNA should be passed through a DNA clean-up column, such as Qiagen
MinElute PCR Purification Kit (part no. 28004), to clean the DNA and minimize the final recovery
volume for a more concentrated DNA sample. To perform the microarray analysis, microgram
quantities of DNA will be needed. Since the amount of DNA recovered from the MethylCollector
Ultra kit depends on the global methylation status of the sample, it may be necessary to pool
multiple samples together or to perform whole genome amplification on the recovered material
in order to obtain microgram quantities. Once microgram quantities of DNA have been obtained,
the MethylCollector Ultra DNA can be labeled with Cy5 and the input DNA can be labeled with
Cy3 for hybridization to the microarray.

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16

Whole genome amplification
Active Motif’s GenoMatrix™ Whole Genome Amplification Kit (Catalog No. 58001) has been successfully validated for use with the GC-rich DNA from the MethylCollector Ultra Kit and shownto maintain the sequence representation of the starting material. It is recommended to start with
500 ng - 1 µg of fragmented DNA per binding reaction, alternatively, multiple small reactions canbe pooled together. 10 ng is the minimum starting material required for the GenoMatrix™ Whole
Genome Amplification Kit.
To amplify DNA without using the GenoMatrix™ Whole Genome Amplification Kit, a Ligated Mediated PCR will need to be performed. This will involve blunt ending the enriched, purified DNA,ligating blunt adaptors and PCR amplifying for 14-20 cycles. Amplicons produced from this method
are generally between 300-500 bp due to a bias for smaller PCR products. The ligation reaction
also has a tendency to be less efficient. However, after amplification you should obtain the microgram quantities of DNA needed for microarray analysis.
Non-amplification
If whole genome amplification will not be performed, multiple MethylCollector Ultra reactions
will need to be combined in order to achieve microgram yields. It may be necessary to combine 10
or more separate binding reactions from the same sample material.
Bisulfite Conversion
Active Motif’s MethylDetector Bisulfite Modification Kit (Catalog No. 55001) can be used toperform DNA conversion with bisulfite. The MethylDetector Kit works in the range of 200 pg to 2
µg of DNA, although special considerations need to be taken when working with samples containing less than 100 ng DNA. Following bisulfite conversion, DNA is purified and cloned into a DNA
vector of choice. Cloned DNA is then transformed into competent bacterial cells, such as Active
Motif’s RapidTrans TAM 1 (Catalog No. 11096) and colonies are screened for positive clones. Plasmid
DNA is isolated from the positive clones and submitted for sequencing. A comparison of bisulfite
converted sequence data against the unconverted DNA sequence will provide the number ofmethylation sites for the region of interest.

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17Section B. PCR Analysis
PCR Primer Design
MethylCollector Ultra includes PCR primers for use with the provided Mse I digested human, malegenomic DNA. If possible, real time PCR is recommended for analysis of DNA isolated with MethylCollector Ultra. To design primers specific to the CpG region of interest in your sample, please
follow the recommendations below.
Primer design considerations
i. Primers should flank the CpG region of interest and produce an amplicon between
100-350 bp in length for endpoint PCR or an amplicon of 100-150 bp for real time PCR.
The CpG region needs to contain at least five methylated CpGs.
ii.

Each primer should be approximately 18-22 nucleotides long, contain 50% GC content
and have a Tm between 55°C-60°C.

iii.

Restriction-digested DNA: PCR primer pairs should amplify a restriction fragment (orportion of a restriction fragment) that contains a CpG-rich region of interest. Eachamplicon must also be free of internal sites for the restriction enzyme.

iv.

Sonicated DNA: PCR primers should flank the CpG-containing region of interest andthe amplicon should not contain any CpG-dinucleotides that are outside of this region.
This will minimize amplification of fragments that are isolated as a result of methylated
CpGs that are near, but not within, the CpG-rich region of interest.

v.

PCR primers should be designed with the aid of a reliable primer design computer
program (e.g., http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Due to thetechnical limitations of PCR, it is sometimes necessary to design more than one primer
pair for a given fragment of interest.

vi.

Potential primer pairs can be evaluated via computer simulation using a program such
as UCSC Genome Browser (http://genome.ucsc.edu/) to ensure the primers selected
will produce a single amplicon in a CpG region of the species being amplified.

Determining Primer Efficiency
It is advised to determine the efficiency of the PCR primers being used. Primers with an efficiency
less than 90% will have poor reproducibility. To calculate primer efficiency:

Primer efficiency (%) = [10(-1/slope)-1] x 100%
To obtain the slope value, follow the instructions for generating and graphing a standard
curve in the Data Analysis and Use of Input DNAs section on page 21. Use the slope of theplotted standard curve in the primer efficiency equation above.

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18

Endpoint PCR Analysis
A typical endpoint PCR protocol example follows below. This protocol was optimized for thecontrol samples. For each new set of primers amplifying the promoter region of interest, the PCR
conditions have to be optimized carefully (optimal Tm, number of cycles, etc.).
1.

For one PCR reaction:
Reagent

One rxn

Sterile water

11.8 µl

10X PCR Buffer

2.5 µl

10X PCR compatible loading dye

2.5 µldNTP mixture (5 mM each dNTP)

1 µl

Forward Primer* (5 pmol/µl)

1 µl

Reverse Primer* (5 pmol/µl)

1 µl

Taq (5 U/µl)

0.2 µlDNA sample (eluted or Input)

5 µl(Optional) 1 M Betaine can be added to PCR reactions as an enhancer to improve PCR results
Total Volume

25 µl* The provided PCR Primer Mixes contain both Forward and Reverse primers for use with theprovided control DNA. Use 2 µl of the PCR primer Mix in the PCR protocol described above.
The Xist PCR primer mix has been optimized for use with low salt binding conditions, while
NBR2 and GAPDH primer mixes can be used with either low or high salt binding conditions.
2.

Place tubes in a PCR thermocycler and program as below:

		3.

94°C for 3 minutes(94°C for 20 seconds, 55°C for 30 seconds, 72°C for 30 seconds) for 36 cycles
Hold at 4°C

Endpoint PCR can be analyzed by agarose gel electrophoresis. Run reactions by loading 10 µlfrom each of the PCRs on a thin 3% agarose gel at 100 V for 40 minutes in parallel with anappropriate DNA ladder. Post-stain the gel with 1 µg/ml ethidium bromide in 1X TAE buffer
for 20 minutes. Observe gel under UV.
GAPDH Glyceraldehyde-3-phosphate dehydrogenase should be unmethylated in the control human, male genomic DNA and is not expected to produce a 69 base pair PCR
product in the eluted DNA fraction.
Xist

X inactive specific transcript is methylated in the control human, male genomic
DNA and will produce a 178 base pair PCR product with the eluted fraction.

NBR2

Neighbor of BRCA1 gene 2 is methylated in the control human, male genomic DNA
and is expected to produce a 103 base pair PCR product with the eluted fraction.

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19

Real Time PCR Analysis
This is an example PCR reaction. Please follow the specific instructions for your real time PCR
instrument.
1.

For one PCR Reaction:
Reagent

10 µl PCR reactions

20 µl PCR reactions

Fast SYBR Green master mix

5 µl

10 µl

Forward primer* (5 pmol/µl)

0.5 µl1 µl

Reverse primer* (5 pmol/µl)

0.5 µl1 µl

Sterile water

1 µl

3 µl

DNA sample (eluted or Input)

3 µl

5 µl

Total volume

10 µl

20 µl

* The provided PCR Primer Mixes contain both Forward and Reverse primers for use with theprovided control DNA. Use 1 µl of the PCR Primer Mix in the 10 µl reaction or 2 µl of the PCRprimer mix in the 20 µl reaction for the PCR protocol described above. The Xist PCR primer
mix has been optimized for use with low salt binding conditions, while NBR2 and GAPDH
primer mixes can be used with either low or high salt binding conditions.
Note:
2.

Place tubes in a Real Time PCR instrument and program as below. The amplification conditions should be optimized for each target locus and PCR instrument. A suggested starting
point is:		3.

It is recommended to prepare triplicates of each sample and Input reaction.
Input DNA should be tested at 0.01, 0.1, 1 and 10 ng/µl to obtain a standard curve.

95˚C for 2 minutes(95˚C for 10 seconds, 60˚C for 30 seconds) for 40 cycles

Analyze the results. Data analysis varies depending on the instrument used. Obtain thestandard curve from the Input samples. Use the standard curve to quantify the DNA in eachsample.
GAPDH Glyceraldehyde-3-phosphate dehydrogenase should be unmethylated in the control human, male genomic DNA and will not amplify in the eluted fraction.
Xist

X inactive specific transcript is a methylated promoter in the control human, malegenomic DNA and will amplify early in the eluted fraction.

NBR2

Neighbor of BRCA1 gene 2 is a methylated promoter in the control human, malegenomic DNA and will amplify early in the eluted fraction.

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20

Data Analysis and Use of Input DNAs
Methylated DNA isolated using MethylCollector Ultra is usually analyzed by PCR amplification of
the loci of interest. However, if the goal is to compare the methylation status of particular lociin different DNA samples, it is essential that MethylCollector Ultra be performed on the sameamount of each DNA sample. Thus, DNA samples should be carefully quantified before use. In
addition, Input DNA should be prepared for each of the different DNA samples (see Step 2. No. 4
in the Protocol) to clearly indicate the relative concentrations of the DNA samples.
For real time PCR, generating a standard curve using the input DNA enables accurate determination of the enriched DNA concentration.
1.

Produce a standard curve at 10, 1, 0.1 and 0.01 ng/µl in triplicate using the Input DNA from
Step2, No. 4. We recommend running a standard curve every time MethylCollector Ultra isperformed. However, if the primer efficiency has been determined to be greater than 90%
according to the calculations on page 18, the standard curve can be generated once andstored for future use with the same DNA sample.

2.

Run each sample with the appropriate DNA standard (i.e. prepare a separate standard curvefor each cell line or species tested).

3.

Every gene will generate a different amplification profile. CT = Threshold Cycle or cycle number where the signal exceeds the background threshold level. CT values should be plottedfor each gene to create a linear regression plot.

4.

Plot CT versus log DNA concentration. See Figure 4 below.
Standard Curve
32

30

CT

28

26

24

22

20
0.001 0.003

0.01

0.03

0.1

0.3

1

3

10

30

100

DNA Concentration (ng/µl)

Figure 4: Example standard curve linear regression plot.
A standard curve for the human, male genomic DNA is provided as a reference only. Input DNA was tested in triplicate andplotted against the CT value. A new standard curve should be generated each time the assay is performed.

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21

5.

Using the CT value of the sample, extrapolate the DNA concentration of the sample DNA using the standard curve plot. To determine the amount of enriched DNA in the sample simply
multiple the DNA concentration by the volume of enriched DNA.

6.

Calculate the percent enrichment. Use the sample DNA quantity calculated above andcompare it with the amount of DNA used in the initial binding reaction (Step 2, No. 6).

		Amount of enriched Sample DNA material
x 100%
Enrichment (%) =		Amount of fragmented DNA used in the binding reaction
7.

Optional: Calculate the fold enrichment of methylated DNA. If the unbound material wascollected in Step 3, No.1 and purified for analysis in PCR, the eluted samples can be comparedto the unbound samples for the same locus in order to determine the fold enrichment.

		
Fold enrichment =		

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Sample DNA concentration in the eluted fraction
Sample DNA concentration in the unbound fraction

22Section C. Use of Magnetic Beads and Included Bar Magnet
Caution: The included neodymium bar magnet is extremely powerful and is easily broken if
handled incorrectly.
1.

The magnet should be stored in the provided tube.

2.

Be careful when working near metal objects or surfaces. A free magnet will jump great
distances onto nearby metal surfaces with surprising speed. This can break the magnet.

3.

If the magnet becomes attached to a flat metal surface, it should be removed by sliding it
off the edge of surface. The magnet may be broken if you attempt to pull one end awayfrom the metal.

Assembly of Magnetic Stands
The provided Mini Glue Dots can be used to attach the bar magnet to an empty tip box to create
an effective magnet stand.

Creating a magnetic stand for 8-well PCR strips:
Note:
8-well strip tubes for use with standard 96-well PCR cyclers are appropriate.
1.

Remove the covering tape from one side of two glue dots.

2.

Place a strip of PCR tubes in the wells of an empty tip box (200 µl tips) and place the magnet
directly against the tubes. This is the way the magnet will be positioned when the glue dots
are used to affix it to the box.

3.

Attach the glue dots on the bar magnet (the uncovered face of the dot is placed on themagnet) as shown below.

4.

Remove the covering tape from the exposed side of the glue dots. Fix the magnet to the tip
box so that it is against the PCR tubes. The magnetic stand is now ready for use.
Note:

Familiarize yourself with using the magnetic stand before performing with PCR
tubes for the first time. Add 5 µl of magnetic beads to 100 µl High Salt Binding Buffer in one tube of an 8-well strip of PCR tubes. Use this tube with theassembled bar magnet stand to become familiar with use of the beads andwww.activemotif.com

23

magnet. It is difficult to re-suspend the beads if the tubes are directly adjacentto the magnet, so it is usually best to move the tubes away from the magnet forresuspension.
Washing should be performed as follows:a. Place the tubes in the rack against the magnet and allow the beads to be pinned to theside of the tube, as shown below.

b.

Remove supernatant with a 200 µl pipetteman or a 200 µl eight-channel pipetteman.

c.

Move the tube strip into a row that is not adjacent to the magnet.

d.

Add wash buffer and pipette up and down to fully resuspend the beads. Ensure that aminimal amount of beads cling to the tips when the resuspension is complete.

e.

Repeat steps a-d until desired washing steps are complete.

Centrifugation of 8-well PCR strip tubes:
When working with 8-well PCR strip tubes, it may be desirable to centrifuge the tubes to collect
the liquid and beads from the inside of the caps. This is easily accomplished in a centrifuge fittedwith adaptors for spinning microtiter plates. In this case, a standard 96-well plate can be placedin the adaptor to hold the tubes in place. Take care to ensure the rotor is balanced (e.g., place a
microtiter plate and tubes of appropriate mass in the rotor’s opposing 96-well plate adaptor). Spinthe plates briefly to let the rotor reach a speed of 1000 x g before allowing the rotor to stop.
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24Section D. Troubleshooting Guide
Problem/question

Recommendation

The target DNA fragment
has less than 5 methylated
CpGs.

The provided Binding Buffers are optimal for efficient capture of DNA fragments thathave five or more methylated CpGs. MethylCollector™ Ultra is not recommended for
isolation of DNA fragments containing less than 5 methylated CpGs.

Little or no enrichmentof methylated DNA in thebound fraction

The binding conditions may be too stringent. Try using the Low Salt Binding Buffer to set
up binding reactions for that particular target.

PCR amplification

It has been determined that using a hot-start polymerase (i.e. Phusion™ from NEB) insteadof a classic Taq polymerase may increase the sensitivity of the assay.
Selection of an appropriate qPCR master mix is important to achieve good amplification.
Since all commercially available mixes have different compositions regarding chemicalenhancers or inhibitors for non-specific amplification, results may vary depending on themaster mix used.
The addition of 1 M Betaine to endpoint PCR reactions can be used to enhance the PCR
and improve the results. Betaine lowers the melting temperature of GC-rich regions making them comparable to AT regions.

Storage of DNA

Once DNA is prepared using MethylCollector Ultra, samples may be stored at -20°C prior
to PCR analysis. However, we recommend heating the frozen material to 37°C for
10 minutes before use in PCR, as heat-treatment releases any DNA bound to the tube
during storage.

Can I use 3 M sodium
acetate, pH 5.2 instead of
5 M ammonium acetate in
the ethanol precipitation?

Yes, 3 M sodium acetate, pH 5.2 can be used at 1/10th sample volume along with 2 sample
volumes of 100% ethanol during the precipitation step. However, we have noticed thatthe ammonium acetate precipitation had better yield of recovery than the sodium
acetate in a direct comparison of several samples.

Should I use Restriction
Digest or Sonication to
fragment my DNA?

Restriction Digest is very precise and reproducible, however, the DNA must be well purified and analysis of several loci may also require use of different enzymes. In addition, theregion of interest may not be flanked by suitable restriction sites and single-nucleotide
polymorphisms (SNPs) between different cell types may confound results.
In contrast, Sonication is random, which enables analysis of many loci simultaneously(microarray), but it may not be possible to shear DNA small enough to isolate CpG islandsof interest. In addition, results may vary from shearing to shearing depending on sonicatorused. Also it is difficult to prepare DNA from a small number of cells.

Heat inactivation or
removal of restriction
enzyme used to fragment
DNA

After restriction digest, we recommend that samples be treated for 20 minutes at
65°C. Some enzymes (such as Mse I) will be inactivated by this treatment, while thosethat are not will be forced off the DNA. In most cases (even when using enzymes thatare not heat-inactivated), DNA treated in this fashion should be suitable for use in the
MethylCollector Ultra protocol. In some situations (e.g., when the DNA used in a digest
is contaminated with cellular proteins or when a large amount of restriction enzyme isrequired for the digest) it may be desirable to purify the digested DNA by purification
columns or through phenol extraction/ethanol precipitation.

10X PCR Loading Dye

If PCR is performed using the 10X PCR Loading Dye provided, it is not necessary to add
additional loading dye to the samples before running samples on agarose gel.

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25Section E. Related Products
DNA Methylation		

FormatCatalog No.

MethylDetector™		
MethylCollector™ Ultra		
UnMethylCollector™		
Hydroxymethyl Collector		
MeDIP		hMeDIP		
DNMT Activity / Inhibition Assay		
Methylated DNA Standard Kit		
5-Carboxylcytosine DNA Standard Kit		
Fully Methylated Jurkat DNA		
Recombinant Tet1 protein		b-Glucosyltransferase enzyme		
PvuRts1I restriction enzyme		

50 rxns
30 rxns
30 rxns
25 rxns
10 rxns
10 rxns
1 x 96 rxns
3 x 2.5 µg
0.5 µg
10 µg
25 µg
500 Units
50 Units

55001
55005
55004
55013
55009
55010
55006
55008
55014
55003
31363
55012
55011

Whole Genome Amplification		

FormatCatalog No.

GenoMatrix™ Whole Genome Amplification Kit		

50 rxns

58001

Antibodies

Application

FormatCatalog No.

DB
DB, IF
DB, IF
DB, IF, IHC, MeDIP
DB, MeDIP
FACS, IHC, IP, MeDIP
DB, MeDIP
ChIP, IHC, IP, WB
WB
ChIP, IF, IHC, WB
ChIP, IF, IP, WB
WB
WB
WB
WB
WB
WB

100 µg100 µl100 µl100 µl100 µg50 µg100 µg100 µg100 µg100 µg100 µg100 µl100 µg100 µg100 µg100 µg100 µg

61111
61225
61223
39769
39999
39649
61255
39204
39205
39206
39207
39907
39215
40965
39216
39217
39218

3-Methylcytosine (3-mC) pAb
5-Carboxylcytosine (5-caC) pAb
5-Formylcytosine (5-fC) pAb
5-Hydroxymethylcytosine pAb
5-Hydroxymethylcytosine mAb
5-Methylcytosine mAb
5-Methylcytosine pAb
DNMT1 mouse mAb
DNMT2 rabbit pAb
DNMT3A mouse mAb
DNMT3B mouse mAb
DNMT3L rabbit pAb
MBD1 mouse mAb
MBD2 mouse mAb
MBD3 mouse mAb
MBD4 mouse mAb
MeCP2 rabbit pAb

Active Motif also offers a growing list of application validated antibodies, including antibodies
for histones and histone modifications, transcription factor antibodies, DNA methylation-related
antibodies and ChIP validated antibodies. For a complete list go to www.activemotif.com/abs
Histone Purification		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		

10 rxns
20 rxns

40025
40026

Chromatin Assembly		

FormatCatalog No.

Chromatin Assembly Kit		
HeLa Core Histones		

10 rxns
36 µg

53500
53501

www.activemotif.com

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Histone Acetyltransferase and Deacetylase Activity

FormatCatalog No.

HAT Assay Kit (Fluorescent)		
Recombinant p300 protein, catalytic domain		
HDAC Assay Kit (Fluorescent)		
HDAC Assay Kit (Colorimetric)		

1 x 96 rxns
5 µg
1 x 96 rxns
1 x 96 rxns

56100
31205
56200
56210

ChIP-IT™ Kits		

FormatCatalog No.

ChIP-IT™ Express		ChIP-IT™ Express Enzymatic		
ChIP-IT™ Express HT		
ChIP-IT™ Protein G Magnetic Beads		
Re-ChIP-IT™		
ChIP-IT™		
ChIP-IT™ w/o controls		
ChIP-IT™ Shearing Kit		
ChIP-IT™ Enzymatic 		
ChIP-IT™ Enzymatic w/o controls		
Enzymatic Shearing Kit		
Salmon Sperm DNA/Protein G agarose		
ChIP-IT™ Control Kit – Human		
ChIP-IT™ Control Kit – Mouse		ChIP-IT™ Control Kit – Rat		
Ready-to-ChIP HeLa Chromatin		
Ready-to-ChIP Hep G2 Chromatin		
Ready-to-ChIP K-562 Chromatin		
Ready-to-ChIP NIH/3T3 Chromatin		

25 rxns
25 rxns
96 rxns
25 rxns
25 rxns
25 rxns
25 rxns
10 rxns
25 rxns
25 rxns
10 rxns
25 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns

53008
53009
53018
53014
53016
53001
53004
53002
53006
53007
53005
53003
53010
53011
53012
53015
53019
53020
53021

Recombinant Methylated Histones		

FormatCatalog No.

Recombinant Histone H2A		
Recombinant Histone H3 (C110A)		
Recombinant Histone H3 monomethyl Lys4		
Recombinant Histone H3 dimethyl Lys4		
Recombinant Histone H3 trimethyl Lys4		
Recombinant Histone H3 monomethyl Lys9		
Recombinant Histone H3 dimethyl Lys9		
Recombinant Histone H3 trimethyl Lys9		
Recombinant Histone H3 monomethyl Lys27		
Recombinant Histone H3 dimethyl Lys27		
Recombinant Histone H3 trimethyl Lys27		
Recombinant Histone H3 monomethyl Lys36		
Recombinant Histone H3 dimethyl Lys36		
Recombinant Histone H3 trimethyl Lys36		
Recombinant Histone H3 monomethyl Lys79		
Recombinant Histone H3 dimethyl Lys79		
Recombinant Histone H3 trimethyl Lys79		
Recombinant Histone H4		
Recombinant Histone H4 monomethyl Lys20		
Recombinant Histone H4 dimethyl Lys20		
Recombinant Histone H4 trimethyl Lys20		

50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg

31251
31207
31208
31209
31210
31211
31212
31213
31214
31215
31216
31217
31218
31219
31220
31221
31222
31223
31224
31225
31226

www.activemotif.com

27

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter andnotification of our upcoming products• Share your ideas and results with us• View our job opportunities
Don’t forget to bookmark our site for easy reference!
www.activemotif.com

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