MethylCollector™ MBD Capture Kit(version A1)

Catalog No. 55026

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
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Telephone: 760 431 1263
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Telephone: (86)-21-20926090
Cell Phone: 18521362870
Copyright 2022 Active Motif, Inc.

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Information in this manual is subject to change without notice and does not constitutea 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, in whole or in part, without the prior written consent of Active Motif, Inc. This
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The manufacturer of this documentation is Active Motif, Inc.
© 2022 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 theirwww.activemotif.com

TABLE OF CONTENTS

Page

Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Fragmentation of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Example Fragmentation Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
MethylCollector MBD Capture Protocol  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Appendix
Section A. Downstream Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Section B. PCR Analysis
		
PCR Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  15
		Endpoint PCR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
		Real Time PCR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  17
		
Data Analysis and Use of Inputs . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Section C. Use of Magnetic Beads and Included Bar Magnet . . . . . . . . . . . . .  21
Section D. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

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Overview
Active Motif’s MethylCollector MBD Capture Kit provides an efficient method for isolating CpG-methylated DNA from limited amounts of cell or tissue samples. MethylCollector has many powerful applications including enabling researchers to rapidly screen
the methylation status of multiple loci and may be particularly useful for analyzing themethylation levels of candidate genes in tumor tissue or cells. It can also be used todetect changes in DNA methylation in other situations, including normal cellular differentiation and aging.
In the MethylCollector MBD Capture Kit, His-tagged recombinant MBD2b protein specifically binds CpG-methylated DNA fragments that have been prepared by enzymatic
digestion or sonication. These protein-DNA complexes are captured with nickel-coatedmagnetic beads and subsequent wash steps are performed to remove fragments withlittle or no methylation. The methylated DNA is then eluted from the beads in the presence of Proteinase K. Due to the high efficiency of the MethylCollector MBD Capture
protocol and the enormous amplification capability and specificity of PCR, analysisof the methylation status of a specific genomic DNA locus can be performed on DNA
isolated from less than 170 cells cells (~1 ng DNA).

product

formatcatalog no.

MethylCollector™ MBD Capture Kit

30 rxns**

55026**MethylCollector™ MBD Capture Kit provides sufficient reagents to perform
30 reactions with excess 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 protein.

Capture the methylated
DNA/MBD2b complexes withnickel-coated magnetic beads.

methylatedDNA

The purified, methylated
DNA is readyfor PCR analysis.

Elute the bound, methylated
DNA while, simultaneously
degrading the MBD2b protein.

Wash to remove theunmethylated
DNA fragments.

Flow chart of the MethylCollector MBD Capture process.
In MethylCollector MBD Capture, genomic DNA of interest is sheared by either enzymatic digestion orsonication. The sheared DNA is then incubated with a His-tagged recombinant MBD2b protein, which
specifically binds CpG-methylated DNA fragments. These protein-DNA complexes are captured with nickelcoated magnetic beads and washes are then performed to remove fragments with little or no methylation.
The methylated DNA is then eluted from the beads. Real time or endpoint PCR can be performed on the
resulting supernatant using specific primers to amplify the locus of interest. Alternatively library prep andDNA sequencing can be performed in order to analyze whole-genome methylation profiles.

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Kit Performance and Benefits
The MethylCollector MBD Capture Kit is for research use only. Not for use in diagnostic procedures.
Sensitivity: MethylCollector MBD Capture can be performed on 1 ng - 1 µg of fragmented genomic DNA.
Nature of the MethylCollector MBD Capture Assay: MethylCollector MBD Capture isan MBD2b based assay for the 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 the locus of interest with customer designed PCRprimers. Control human, male genomic DNA that was 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 malegenomic DNA. The region amplified by this primer pair is 178 base pairs andcontains 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 boundand eluted from the protein as detected with the NBR2 PCR Primer Mix. This means thatof 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.
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 boundand eluted from the protein as detected with the Xist PCR Primer Mix. This means thatof 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 GAPDH PCR Primer Mix for either low or high salt binding conditions.
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A.

B.

C.

D.

Figure 1: Real time PCR analysis of control PCR primer sets.
MethylCollector MBD Capture was performed using 100 ng of Mse I digested human, male genomic DNA
under both low and high salt conditions. Eluted DNA was purified and analyzed using real time PCR for bothmethylated and unmethylated promoters. A) Amplification plot using the provided NBR2 PCR primer mix withthe unbound and eluted fractions under high salt binding conditions. NBR2 is methylated 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 under low salt binding conditions. 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 unbound and eluted fractions under high salt binding conditions. GAPDH is unmethylated in the control DNA and shows late amplification of the eluted fractions. D) Amplification plot using theprovided GAPDH PCR primer mix with the unbound and eluted fractions under low salt binding conditions.
GAPDH is unmethylated in the control DNA and shows late amplification of the eluted fractions.

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Figure 2: Percent enrichment of MethylCollector MBD Capture binding reactions.
MethylCollector MBD Capture 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 thebinding reaction to produce a percent enrichment. The methylated promoters NBR2 and Xist had greater than
10-fold enrichment of eluted DNA as compared to unbound DNA for the same locus, while the unmethylated
GAPDH promoter was found almost exclusively in the unbound fraction.

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5Kit Components and Storage
Kit components arrive on dry ice. Upon receipt, we recommend storing each component at the temperatures listed in the table below. The magnetic beads can be stored
frozen upon arrival, however, we recommend long-term storage at 4°C. Once themagnetic beads have been thawed, do not re-freeze the beads.
Reagents

Quantity

Storage / Stability

His-MBD2b protein

350 µl-80°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

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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 dots (see Appendix - Section C) or use commercially available stands• Rotisserie shaker• Phenol/chloroform or DNA clean-up kit for purification of DNA such as Active Motif’s Chromatin IP DNA Purification Kit (Catalog No. 58002).
• 5 M Ammonium acetate (see Troubleshooting Guide, Appendix - Section D, fordetails 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 protein 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. DNA can be fragmented by restriction digest or by mechanicalmeans (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 are contained within a particular restriction fragment. This fragment should
be long enough (75 bp or longer) to allow for PCR analysis. Some useful methylationinsensitive restriction enzymes are shown 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 DNA sample will be used for simultaneous analysis of many CpG islands. When the isolated DNA will be analyzed by NGS
sequencing or by microarray methods, or when a CpG region of interest is not flanked
by suitable restriction sites, sonication is recommended. 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 9. We suggest using 4 µg of purified genomic DNA when
performing restriction digestion and 20 µg of purified genomic DNA for sonication.
MethylCollector MBD Capture reactions can be performed on 1ng - 1 µg of fragmented
genomic DNA.
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Example Framentation 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 a commercially available kit or a standard established protocol. The qualityof 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) 10 µl
10X NEB Buffer 4		
10 µl
100X BSA			
1 µl
Mse I (10 U/µl)		
1 µl
dH2O			78 µl
Total volume		
100 µlNote 1:
Note 2:b)c)

The DNA volume may vary depending on its initial concentration.
MethylCollector MBD Capture has been used with as little as 1 ng ofrestriction-digested genomic DNA. As a reference, a human cell contains about 6 picograms DNA; 1 ng of genomic DNA corresponds to 170
cells.
Mix well by pipetting and incubate at 37°C for 2 hours to overnight.
Heat-inactivate Mse I by incubating the reaction mixture at 65°C for 20 minutes. If
using an alternative restriction enzyme that cannot be heat-inactivated, the DNA
can be purified by phenol/chloroform extraction and precipitation, or using a DNA
purification Kit. See “Appendix D. Troubleshooting” on page 25 for comments
about heat-inactivation.
Note 1:
Note 2:For greater accuracy, the digested DNA should be quantified.
This digested DNA should be stored at -20°C until use.

Mechanical fragmentation (sonication)
Restriction enzymes are not always ideal to fragment and isolate the DNA sequences ofinterest. In this case sonication of the genomic DNA is an alternative method.
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 canbe 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 MBD Capture 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, pleaserefer to Appendix A.
2.

Two different binding buffers are included in the kit. For high salt conditions (highstringency reactions) we recommend using High Salt Binding Buffer. For low saltconditions (lower stringency reactions) we recommend using Low Salt Binding
Buffer. It may be necessary to perform experiments with both binding conditions todetermine the optimal conditions for the 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 of Complete 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 of Complete 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 MBD Capture protocol can be performed on a large range of sample DNA amounts (1 ng to1 µg). We recommend 100 ng because it gives robust results without requiring a
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large amount of DNA.
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 thekit, it is recommended 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 thekit, 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 by diluting the DNA 1/4 in dH2O (e.g., 5 µl of 20 ng/µl DNA + 15 µl dH2O to make 5 ng/µl DNA). 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 PCR primers 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 and you may want to try several amounts of
Input DNA to be sure to obtain PCR products from reactions still in thelinear phase of amplification.

5.

Using the PCR tubes provided, fully resuspend magnetic beads by inverting, thenaliquot a 10 µl slurry into each tube. If preparing more than 4 reactions, cap andre-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 toeach PCR tube. Pipet the His-MBD2b protein 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

1 ng-1 µg (10 µl)-

Control Human, male genomic DNA(Mse I digested)

-

5 µl

His-MBD2b protein

10 µl

10 µl

Total Volume

100 µl100 µlNote:

It is recommended to aliquot the provided His-MBD2b protein into
several small fractions for future use. Pipet the His-MBD2b protein up
and down several times to ensure homogeneity before making aliquots.
Store at -80°C.

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8.

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

Step 3: Wash beads
1.

After the capture step is complete, spin the PCR tubes briefly and place tubes on amagnetic 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 enrichmentof methylated DNA, place supernatant in a microcentrifuge tube and set aside at
4°C for DNA clean up in Step 5 of the protocol. Otherwise, remove and discard thesupernatant. To use the magnet provided in the kit, 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 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 the strength of the magnet being used, it may be necessary to remove
the tubes from the magnet and place in a separate rack to fully resuspend thebeads.

d.

Repeat steps a-c one time.

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 tothe 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 toensure the beads 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 DNA can be stored at -20°C. If the DNA isstored at -20°C, it is recommended to be reheated at 37°C for 10 minutes prior touse.
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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 specificdownstream applications.
For more concentrated DNA, use the MinElute PCR Purification Kit (Qiagen part no.
28004) and elute in as low as 10 µl.
• 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 theeluted 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 withoutcollecting 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 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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Appendix
Section A. Downstream Applications
The MBD2b protein used in the MethylCollector MBD Capture 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 protein increases
with the density of methyl CpG dinucleotides. The amount of DNA recovered from the
MethylCollector MBD Capture Kit will depend on the global methylation status of thesample 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 anincrease in methylation at that single site. Alternatively, the same site can be comparedacross different sample types, but it will be necessary to run MethylCollector MBD
Capture on the same amount of each DNA sample and generate a separate standard
curve for each sample.
Next-Gen Sequencing Analysis
To analyze whole-genome methylation profiles, researchers may chose to perform
libray prep and Next-Gen sequencing following the enrichment of methylated DNA withMethylCollector MBD Capture. This process has been validated when DNA enrichedwith MethylCollector MBD Capture is end adapted for use in sequencing. We recommend using a kit like the Active Motif Next Gen DNA Library Kit, cat# 53216, to create
libraries.

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14Section B. PCR Analysis
PCR Primer Design
MethylCollector MBD Capture includes PCR primers for use with the provided Mse I
digested human, male genomic DNA. If possible, real time PCR is recommended foranalysis of DNA isolated with MethylCollector MBD Capture. To design primers specificto 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 ampliconbetween 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 (or portion of a restriction fragment) that contains a CpG-rich region ofinterest. Each amplicon must also be free of internal sites for the restriction
enzyme.
iv.

Sonicated DNA: PCR primers should flank the CpG-containing region ofinterest and the amplicon should not contain any CpG-dinucleotides that areoutside 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 CpGrich region of interest.

v.

PCR primers should be designed with the aid of a reliable primer design
computer program. Due to the technical 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 to ensure the primers selected willproduce 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 anefficiency 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 astandard curve in the Data Analysis and Use of Input DNAs section on page 21. Use
the slope of the plotted standard curve in the primer efficiency equation above.
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Endpoint PCR Analysis
A typical endpoint PCR protocol example follows below. This protocol was optimized
for the control samples. For each new set of primers amplifying the promoter regionof interest, the PCR conditions have to be optimized carefully (optimal Tm, number ofcycles, etc.).
1.

For one PCR reaction:
Reagent

One rxn

Sterile water

11.8 µl

10X PCR Buffer

2.5 µl

10X PCR 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 foruse with the provided 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 withlow salt binding conditions, while NBR2 and GAPDH primer mixes can be used witheither 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 µl from each of the PCRs on a thin 3% agarose gel at 100 V for 60
minutes in parallel with an appropriate 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 ge-

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nomic 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 withthe eluted fraction.

Real Time PCR Analysis
This is an example PCR reaction. Please follow the specific instructions for your realtime 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 foruse with the provided control DNA. Use 1 µl of the PCR Primer Mix in the 10 µl
reaction or 2 µl of the PCR primer 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 astandard 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. Obtainthe standard curve from the Input samples. Use the standard curve to quantify the
DNA in each sample.

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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, male genomic DNA and will amplify early in the eluted fraction.

NBR2

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

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Data Analysis and Use of Input DNAs
Methylated DNA isolated using MethylCollector MBD Capture is usually analyzed by
PCR amplification of the loci of interest. However, if the goal is to compare the methylation status of particular loci in different DNA samples, it is essential that MethylCollector MBD Capture be performed on the same amount 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) toclearly 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 MBD Capture is performed. 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 and stored for future use with the same
DNA sample.

2.

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

3.

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

4.

Plot CT versus log DNA concentration. See Figure 3 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 3: 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 intriplicate and plotted against the CT value. A new standard curve should be generated each time the assay is
performed.
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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
and compare it with the amount of DNA used in the initial binding reaction (Step 2,
No. 6).

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

Optional: Calculate the fold enrichment of methylated DNA. If the unboundmaterial was collected in Step 3, No.1 and purified for analysis in PCR, the eluted
samples can be compared to the unbound samples for the same locus in order todetermine the fold enrichment.

		
Sample DNA concentration in the eluted fraction
Fold enrichment =		Sample DNA concentration in the unbound fractionwww.activemotif.com

20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 breakthe magnet.

3.

If the magnet becomes attached to a flat metal surface, it should be removed bysliding it off the edge of surface. The magnet may be broken if you attempt to pull
one end away from 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
the magnet) 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 readyfor use.
Note:

Familiarize yourself with using the magnetic stand before performing

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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 the assembled bar magnet stand to become familiar withuse of the beads and magnet. It is difficult to re-suspend the beads if
the tubes are directly adjacent to the magnet, so it is usually best to
move the tubes away from the magnet for resuspension.
Washing should be performed as follows:a. Place the tubes in the rack against the magnet and allow the beads to bepinned to the side 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 a minimal 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
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to collect the liquid and beads from the inside of the caps. This is easily accomplishedin a centrifuge fitted with adaptors for spinning microtiter plates. In this case, a standard 96-well plate can be placed in the adaptor to hold the tubes in place. Take careto 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). Spin the plates briefly to let therotor reach a speed of 1000 x g before allowing the rotor to stop.

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23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 fragmentsthat have five or more methylated CpGs. MethylCollector MBD Capture is notrecommended for isolation of DNA fragments containing less than 5 methylated
CpGs.

Little or no enrichmentof methylated DNA in
the bound 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) instead of a classic Taq polymerase may increase the sensitivity of theassay.
Selection of an appropriate qPCR master mix is important to achieve good
amplification. Since all commercially available mixes have different compositions regarding chemical enhancers or inhibitors for non-specific amplification,
results may vary depending on the master 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 MBD Capture, samples may be
stored at -20°C prior to PCR analysis. However, we recommend heating thefrozen 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 insteadof 5 M ammonium
acetate in the ethanol
precipitation?

Yes, 3 M sodium acetate, pH 5.2 can be used at 1/10th sample volume alongwith 2 sample volumes of 100% ethanol during the precipitation step. However,
we have noticed that the ammonium acetate precipitation had better yield ofrecovery 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, the region 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 toisolate CpG islands of interest. In addition, results may vary from shearing toshearing depending on sonicator used. Also it is difficult to prepare DNA from asmall 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 those that are not will be forced off the DNA. In most cases (even whenusing enzymes that are not heat-inactivated), DNA treated in this fashion should
be suitable for use in the MethylCollector MBD Capture protocol. In some situations (e.g., when the DNA used in a digest is contaminated with cellular proteinsor when a large amount of restriction enzyme is required for the digest) it may
be desirable to purify the digested DNA by purification columns or throughphenol 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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24Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of
the numbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150 Toll Free: 877 222 9543
Carlsbad, CA 92008, USA
Direct: 760 431 1263
E-mail: tech_service@activemotif.com
Fax: 760 431 1351
Active Motif Europe
Waterloo Atrium
Drève Richelle 167 – boîte 4
BE-1410 Waterloo, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Active Motif China
889 Wanhangdu Road, Suite 1602B Direct: (86)-21-20926090
Jingan District
Cell Phone: 18521362870
Shanghai, China 200040
E-mail: techchina@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com

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