MethylDetector™
Bisulfite Modification Kit(version B)

Catalog No. 55001

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
104 Avenue Franklin Roosevelt
B-1330 Rixensart, 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
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2010 Active Motif, Inc.

www.activemotif.com

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.
© 2010 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.
www.activemotif.com

TABLE OF CONTENTS

Page

Overview  . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction  . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Traditional Methods to Study DNA Methylation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
MethylDetector Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Section B. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

www.activemotif.com

Overview
Active Motif’s MethylDetector™ Bisulfite Modification Kit simplifies analysis of DNA methylationby providing optimized reagents for performing DNA conversion with bisulfite, plus time-saving
DNA purification columns and positive control PCR primers to validate results.
DNA methylation is a naturally occurring event that affects cell function by altering gene expression. In methylation, a methyl group is added to the fifth-carbon of cytosine in a CpG dinucleotide by DNA methyltransferase. As aberrant methylation is prevalent in many human cancers, andbecause methylation is also involved in embryonic development and cell cycle regulation, muchresearch depends on accurately quantifying DNA methylation. Many DNA methylation analysis
methods begin by using bisulfite to convert unmethylated cytosines to uracils.1, 2 During conversion, methylated cytosines remain unchanged. The DNA is then amplified by PCR and analyzed
by sequencing or restriction digest. A methylation profile of the sample can then be created bycomparing the sequence of the converted DNA to untreated DNA. However, bisulfite conversion
can be technically challenging, and it is desirable to confirm that the process was successful before
costly analysis of samples. The MethylDetector Kit provides optimized conversion reagents, aneasy-to-use protocol and positive control PCR primers that are specific for bisulfite-converted
human DNA. Because these primers produce PCR product only if conversion has occurred, you canconfirm the procedure worked before starting sequencing or other analysis methods.
In the MethylDetector method, DNA of interest is rapidly heat denatured in a thermocycler in thepresence of the bisulfite conversion reagent. The temperature is then lowered and the conversion
reaction is performed. Unlike other methods, MethylDetector does not require an initial alkaline
denaturation step as the conversion reagent includes a DNA denaturant. After DNA conversion,
the sample is added to the included DNA purification columns, and a simple, on-column desulfonation is performed. Ready-to-use DNA is then eluted from the columns. For your convenience,
the included positive control PCR primers can be used to assess the success of the bisulfite
conversion before DNA sequencing because the included primers only anneal to converted humanDNA.

product

formatcatalog no.

MethylDetector™

50 rxns

55001

www.activemotif.com

1Flow Chart of Process;\eXkli\;E8n`k__\Xk]fccfn\[Yp:fem\ij`feI\X^\ek;\jlc]feXk\#
:c\Xe$lgXe[G:I

Flow chart of the MethylDetector™ process.
In MethylDetector, DNA of interest is rapidly heat denatured in a thermocycler in the presence of the bisulfite
conversion reagent. The temperature is then lowered and the conversion reaction is performed in the presence of a
DNA denaturant. After DNA conversion, the sample is added to the included DNA purification columns, and a simple
on-column desulfonation is performed. DNA is then eluted from the column and is ready for use in PCR.

Introduction
Over the last decade, the study of DNA methylation and its role in epigenetic cell signaling has
grown rapidly3-6. Cellular methylation of CpG-dinucleotides, which occurs at the fifth position of
the cytosine 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 unmethylated7. Aberrant hypermethylation of CpG islands and subsequent transcriptional repression is oneof the earliest and most common somatic genome alterations in multiple human cancers8, 9. 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 identified10.
Aberrant methylation of CpG islands thus seems to be a tumor type-specific event9, 11 and current
efforts have concentrated on finding ways to exploit the diagnostic and therapeutic implicationsof these abnormalities12, 13.

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-dinucleotides14. Methylation-sensitive restrictionwww.activemotif.com

2

enzymes have several limitations including that methylation-sensitive restriction merely
informs on the methylation status of the cytosine residues which are recognized by the
restriction enzymes used.
2. Bisulfite conversion: Bisulfite conversion15 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 converted DNA can be sequenced in order to quantify
the level of DNA methylation in the sample. However, bisulfite-based techniques do involve
chemical treatments that can damage DNA and limit throughput. Additionally, PCR primer
design becomes difficult due to reduction in genome complexity after bisulfite treatment.
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 sonication16. 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.

Kit Performance and Benefits
The MethylDetector Kit is for research use only. Not for use in diagnostic procedures.
Sensitivity: MethylDetector can be performed on 200 pg - 2 µg of DNA.
M

1

2

3

4

5

500 bp400 bp300 bp200 bp100 bpFigure 1: Agarose gel analysis of PCR products generated with MethylDetector.
The MethylDetector DNA conversion method was performed in triplicate on human genomic DNA. The kit’s conversion-specific positive control primers were used to perform PCR on the converted DNA (lanes 1-3) and on unconverted DNA(lane 5). Lane 4 is a no DNA control. The absence of PCR product in lane 5 demonstrates the conversion-specificity of
the PCR primers while the PCR product in lanes 1-3 confirms that the DNA conversion was successful.

www.activemotif.com

3Kit Components and Storage
Kit components arrive at room temperature and can be stored at 4ºC prior to first use. Then, we
recommend storing each component at the temperatures listed in the table below:
Reagents

Quantity

Storage / Stability

Conversion Reagent

5 tubes

RT for 6 months

Denaturation Reagent

875 µlRT for 6 months

Hydroquinone

5 x 100 µl

RT for 6 months

Buffer A

1.75 ml

RT for 6 months

Buffer B

1.1 mlRT for 6 months

DNA Binding Buffer

2 x 15 ml

RT for 6 months

DNA Wash Buffer

25 ml

RT for 6 months

DNA Elution Buffer

2.5 mlRT for 6 months

DNA purification columns

50 ea

RT for 6 months

DNA column collection tubes

50 ea

RT for 6 months

p16 Outer Primer Pair

200 µl

-20°C for 6 months

p16 Inner Primer Pair

200 µl

-20°C for 6 months

10X PCR Buffer

1.5 ml

-20°C for 6 months

Additional Materials Required• 0.2 ml PCR tubes and caps
• 100% isopropanol• Microcentrifuge tubes and microcentrifuge• dNTP mixture (5 mM each)• PCR cycler• Taq polymerase (5 U/µl) (Example: New England Biolabs M0267L or GeneSpin STS-T1000)

www.activemotif.com

4

Protocols
Buffer Preparation and Recommendations
Preparation of Conversion Buffer
Five tubes of Conversion Reagent are provided in MethylDetector. Each vial is sufficient for
performing 10 bisulfite conversions. Prepare the Conversion Buffer by resuspending one of theConversion Reagent tubes with 700 µl dH2O, 350 µl Buffer A and 175 µl Denaturation Reagent. Mix
at room temperature for 10 minutes with intermittent vortexing.
NOTE:

This is a saturated solution and it is normal for the Conversion Reagent to notdissolve completely. The Conversion Buffer is stable for 1 week at -20°C.

Preparation of Hydroquinone
Hydroquinone is an alkalizing agent and prevents DNA from strand breakage because of
depurination. Each tube provided can be used for 10 conversion reactions. Prepare a working stockof Hydroquinone by resuspending one tube with 100 µl dH2O. Keep mixture in dark and discard
after use.
Preparation of Desulfonation Buffer
For each reaction, combine 22 µl Buffer B with 88 µl dH2O and 110 µl 100% isopropanol. If thisreagent will not be used immediately, it can be stored for up to one week at -20°C.
Positive control PCR primers
The MethylDetector’s positive control PCR primers are specific for converted human DNA. These
primers amplify a region of the p16 locus and demethylation of this locus has been shown to correlate with up-regulation of the p16INK4A gene in human gastric cancer cell lines17. This locus has
an official symbol of CDKN2A (cyclin-dependent kinase inhibitor 2A) and is located on chromosome 9, location 9p21 in humans. Other aliases include HGNC:1787, ARF, CDK4I, CDKN2, CMM2,
INK4, INK4a, MLM, MTS1, p16, p14, p14ARF, p16, p16INK4, p16INK4a, p19.
Design of PCR primers
PCR primer design is critical for successful analysis of converted DNA. There are several sites thatoffer information on designing methylation PCR primers. The MethPrimer website18: http://
www.urogene.org/methprimer/ is a free site that contains several useful tools for the design ofbisulfite-conversion-based Methylation PCR Primers. In addition, PREMIER Biosoft International
offers the Beacon Designer program http://www.premierbiosoft.com/crm/jsp/com/pbi/crm/
clientside/ProductList.jsp. To ensure effective and specific amplification of the desired target, werecommend that nested PCR primers be used whenever possible.

www.activemotif.com

5

GCAGGTGGGGAGGAGCCCAGTCCTCCTTCCTTGCCAACGCTGGCTCTGGCGAGGGCTGCT|:|||||||||||||:::|||::|::||::|||::||++:|||:|:|||++||||:||:|GTAGGTGGGGAGGAGTTTAGTTTTTTTTTTTTGTTAACGTTGGTTTTGGCGAGGGTTGTT
TCCGGCTGGTGCCCCCGGGGGAGACCCAACCTGGGGCGACTTCAGGGGTGCCACATTCGC|:++|:|||||::::++|||||||:::||::|||||++|:||:|||||||::|:|||++:TTCGGTTGGTGTTTTCGGGGGAGATTTAATTTGGGGCGATTTTAGGGGTGTTATATTCGTTAAGTGCTCGGAGTTAATAGCACCTCCTCCGAGCACTCGCTCACAGCGTCCCCTTGCCTG||||||:|++||||||||||:|::|::|:++||:|:|++:|:|:||++|::::|||::||TAAGTGTTCGGAGTTAATAGTATTTTTTTCGAGTATTCGTTTATAGCGTTTTTTTGTTTGGAAAGATACCGCGGTCCCTCCAGAGGATTTGAGGGACAGGGTCGGAGGGGGCTCTTCCGC||||||||:++++||:::|::|||||||||||||||:|||||++|||||||:|:||:++:GAAAGATATCGCGGTTTTTTTAGAGGATTTGAGGGATAGGGTCGGAGGGGGTTTTTTCGTCAGCACCGGAGGAAGAAAGAGGAGGGGCTGGCTGGTCACCAGA:||:|:++|||||||||||||||||||:|||:||||:|::|||TAGTATCGGAGGAAGAAAGAGGAGGGGTTGGTTGGTTATTAGA

Figure 2: MethylDetector’s PCR primers are specific for only converted DNA
The figure above depicts the region of the human p16 locus that is used as the positive control in MethylDetector. The
top row is the original, unconverted sequence, while the lower row is the bisulfite modified sequence. For this display, it
is assumed that all CpG sites are methylated. As depicted in the figure, the p16 outer primers and the p16 inner forward
primer are conversion specific because they anneal with no mismatches to the converted sequence, while each primer has
several mismatches when compared with the unconverted sequence. The p16 inner reverse primer matches 100% with boththe converted and the unconverted DNA.
Legend++ Indicates a CpG site, : Indicates Non-CpG ‘C’ converted to ‘T’
The p16 outer forward primer is in bold. The p16 outer reverse primer anneals to the sequence and is in italics. The p16 inner
forward primer is underlined and the p16 inner reverse primer anneals to the sequence and is bold and underlined. The p16
inner reverse primer and the p16 outer reverse primer overlap slightly.
The outer primer sequences are:
p16 Forward outer: GTAGGTGGGGAGGAGTTTAGTT
p16 Reverse outer: TCTAATAACCAACCAACCCCTCC
The outer pair flanks a 283-bp region of the p16 locus.
The Inner primer sequences are:
p16 Forward inner: GGGGGAGATTTAATTTGG
p16 Reverse inner: CCCTCCTCTTTCTTCCTC

www.activemotif.com

6

MethylDetector Protocol
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
The amount of DNA used in MethylDetector should range from 200 pg to 2 µg, with 500 ng to 2
µg optimal. Please see the Appendix A: Troubleshooting section for suggestions when using lessthan 100 ng of DNA. In addition, the positive control PCR primers are designed to anneal to converted human DNA. If you plan to perform the positive control reactions, it is recommended thatyou perform the conversion reaction with 500 ng of human DNA.
Step 1: Conversion Reaction
1. Prepare Conversion Buffer and Hydroquinone as described in the Buffer Preparation section
on page 4.
2.

Set up a conversion reaction for each sample by adding the reagents in the order listed
below into PCR tubes. Mix well by pipetting. The final volume should be 140 µl. If using lessthan 13 µl of DNA, adjust the volume with water to a final volume of 140 µl. Keep hydroquinone away from light.
Reagent

Quantity

DNA

13 µl

dH2O

X µl

Hydroquinone

7 µl

Conversion Buffer

120 µlTotal Volume

140 µl3.

Place tubes in thermocycler and set the program to start with an initial melt at 94°C for 3
minutes, then a 50 °C conversion for 5 hours, followed by a hold at 4°C. To prevent evaporation of samples, use the heated lid or overlay reactions with mineral oil.

4.

Remove tubes and continue with Step 2 below. The protocol can also be stopped here andDNA samples can be kept at 4°C in the dark for up to 5 days.

Step 2: On-column desulfonation and DNA purification
1. Prepare Desulfonation Buffer as described in the Buffer Preparation section on page 4.
2.

For each conversion reaction, aliquot 500 µl DNA Binding Buffer into a 1.5 ml microcentrifuge
tube. Add the entire conversion reaction mixture to the DNA Binding Buffer in the tube andvortex.

3.

Remove the desired number of DNA purification columns and place each one in a collection
tube.

4.

Pipet each DNA Binding Buffer/conversion reaction mixture into one of the DNA Purification
columns and spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection
tube and place the column back on the collection tube.
www.activemotif.com

7

5.

Add 200 µl DNA Wash Buffer to each column and spin at 10,000 rpm for 30 seconds in a
microcentrifuge.

6.

Add 200 µl Desulfonation Buffer to each column and incubate 20 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection tube andplace the column back on the collection tube.

7.

Add 200 µl DNA Wash Buffer to each column and spin at 10,000 rpm for 30 seconds in a
microcentrifuge. Remove the column and place in a new 1.5 ml microcentrifuge tube.

8.

Add 50 µl DNA Elution Buffer directly to the filter of the column.

9.

Incubate 3 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. The eluate will contain the converted DNA.

10. The eluted DNA is now ready for analysis by using methylation-specific PCR or PCR amplification followed by sequencing.
OPTIONAL - Use of Positive Control p16 Primers to confirm conversion
Step 3: Initial PCR using p16 Outer primers
Set up a PCR reaction by using the p16 Outer Primer Pair. Three reactions are performed: one withconverted DNA as template, one with dH2O as template (the no DNA control) and one with 40 ng
unconverted DNA as template.
1.

Label PCR tubes 1-3 and place in a PCR tube rack on ice.

2.

Make a 10 ng/µl dilution of uncut, unconverted human genomic DNA (the same DNA thatwas used for the conversion reaction).

3.

Prepare p16 outer PCR cocktail in 1.5 ml microcentrifuge tubes as described below. DNA templates will be added later directly to the PCR tubes. Combine components on ice and mix by
vortexing. Four reactions are prepared to account for any loss during pipetting.
Reagent

One reaction

4 reactions

dH2O

19.3 µl77.2 µl

p16 Outer Primer Pair

3.0 µl12 µl

dNTP mixture (10 mM each dNTP)

0.5 µl

2.0 µl

10X PCR Buffer

3.0 µl12 µl

Taq (5 U/µl)

0.2 µl

0.8 µlTotal Volume(Not including DNA template)

26 µl

104 µlNote:

10X PCR buffer provided in the kit is for your convenience. If preferred, use the
10X PCR buffer provided by the manufacturer of the Taq polymerase enzyme
being used in the PCR reaction.

www.activemotif.com

8

4.

Aliquot 26 µl of this PCR cocktail to the PCR tubes 1-3 on ice.

5.

Add template DNA to the PCR tubes as indicated below and mix. Ensure that the PCR reaction is on the bottom of the tube and centrifuge tubes if necessary.
PCR rxn #6.

PCR cocktail

DNA template

dH2O

1

26 µl

4 µl converted DNA

0

2

26 µl

0

4 µl

3

26 µl

4 µl 10 ng/µl unconverted DNA

0

Place tubes in PCR cycler and program the thermocycler as below. (To prevent evaporation ofsamples, use the heated lid or overlay reactions with mineral oil.)
94°C for 3 minutes(94°C for 30 seconds, 50°C for 30 seconds, 72°C for 30 seconds) for 20 cycles
72°C for 4 minutes
Hold at 4°C

Step 4: Nested PCR using p16 Inner primers
1. After the initial PCR is complete, label three PCR tubes 1-3 and place on ice.
2.

Prepare p16 inner PCR cocktail in 1.5 ml microcentrifuge tubes as below. DNA templates will
be added later directly to the PCR tubes. Combine components on ice and mix by vortexing.
Four reactions are prepared to account for any loss during pipetting.
Reagent

One reaction

4 reactions

dH2O

19.3 µl77.2 µl

p16 Inner Primer Pair

3.0 µl12 µl

dNTP mixture (10 mM each dNTP)

0.5 µl

2.0 µl

10X PCR Buffer

3.0 µl12 µl

Taq (5 U/µl)

0.2 µl

0.8 µlTotal Volume(Not including DNA template)

26 µl

104 µl3.

Aliquot 26 µl of this PCR cocktail to the PCR tubes 1-3 on ice.

4.

Add 4 µl of the Outer PCR DNA to the appropriate Inner PCR tube and mix as in table below.
Ensure that the PCR reaction is on the bottom of the tube and centrifuge tubes if necessary.
PCR rxn #PCR Cocktailµl for 3 ChIP rxns

1

26 µl

4 µl Outer PCR1

2

26 µl

4 µl Outer PCR2

3

26 µl

4 µl Outer PCR3

www.activemotif.com

9

5.

Place tubes in PCR cycler and program the thermocycler as below. (To prevent evaporation ofsamples, use the heated lid or overlay reactions with mineral oil.)
94°C for 3 minutes(94°C for 30 seconds, 50°C for 30 seconds, 72°C for 30 seconds) for 30 cycles
72°C for 10 minutes
Hold at 4°C

Step 5: Agarose Gel Analysis of PCR products
1. Pour a 2.5% low molecular weight agarose gel in 1X TAE.
2.

Add 10 µl from each of the p16 inner PCRs to 2 µl 6X DNA loading dye and load sample on agel. Use a 100 or 50 bp ladder. Run the gel until the marker is near the bottom of the gel andthe markers are well separated. Only the lane containing the converted DNA should show astrong band at 190 bp. See sample data on page 3.

Step 6: DNA clean up
Prior to cloning it is necessary to clean up the DNA. Use the following protocol to perform aphenol/chloroform extraction followed by ethanol precipitation. Alternatively, DNA can be purified using columns such as QIAquick PCR purification kit (Qiagen part no. 28104). Elute in 30-50 µl
volume. If more than one band is amplified by the nested PCR reaction in Step 4, the DNA band of
the expected size should be gel-purified using a gel extraction kit such as QIAEX II gel extraction
kit (Qiagen part no. 20021).
1.

To the remaining PCR reaction from Step 4, add the necessary amount of sterile water tomake the total volume 100 µl.

1.

Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v/v) to the sample.

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 ng/µl)
1/10 sample volume of 3 M sodium acetate pH 5.5
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.

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

10

11.

Carefully discard the supernatant without disturbing the pellet.

12.

Air-dry the pellet for 5-10 minutes (do not completely dry the pellet).

13.

Resuspend the DNA pellet in 30 µl sterile DNase-free water.

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 to ensure DNA does not
stick to the walls of the tube.

Step 7: DNA cloning
1. The cleaned PCR product can be cloned into a DNA vector of choice according to the instructions of the manufacturer. If possible, plasmids containing the LacZ gene for blue/white
screening are recommended.
2.

Transform competent bacterial cells, such as RapidTrans™ TAM 1 (Active Motif Cat. No.
11096), with 2 µl of the ligation reaction. Follow the appropriate protocol for the competentcells used. The RapidTrans™ TAM 1 chemically competent cell protocol is listed below as an
example.
a.

Remove required number of transformation reaction tubes from –80°C storage andplace on ice to thaw.

b.

Add 1-5 μl of plasmid DNA/ligation to thawed cells. Mix by tapping tubes gently andreplace on ice immediately. Do not mix by vortexing or pipetting. Do not add morethan 5 μl (10% of competent cell volume) to reaction.

c.

Incubate transformation reactions on ice for 30 minutes.

d.

Heat-shock the tubes by immersing in a 42°C water bath for exactly 30 seconds.

e.

Replace transformation reactions on ice for 2 minutes.

f.

Aseptically add 250 μl SOC medium to each reaction.

g.

Incubate tubes at 37°C for 1 hour with shaking at 225-250 rpm.

h.

Using a sterile spreader, plate out 20-200 μl of each transformation on pre-warmed
LB agar plates. If using blue/white screening, ensure that the selective plate contains
X-Gal. For preparation of X-gal Stock solution and plates see note below.

Note:

3.

Make a 40 mg/ml solution of X-gal in dimethylformamide (DMF). Use a glass or
polypropylene tube and wrap in aluminum foil to prevent damage by light. Store
at –20°C. Plate 40 μl of X-gal stock solution on top of the LB agar and let standfor approximately 1 hour. Alternatively, add the X-gal to the molten LB agar prior
to pouring plates.

i.

Allow plates to completely absorb any excess media.

j.

Incubate inverted plates overnight at 37°C.

Select 10 colonies for screening. Select white colonies if using blue/white screening as theexpression of LacZ has been interrupted with the insertion of the ligation product.
www.activemotif.com

11

4.

Isolate plasmid DNA of the 10 selected colonies using any commercially available kit or
published protocol.

5.

Analyze the isolated DNA using restriction enzyme analysis to confirm the presence of thecorrect insert.

6.

Submit DNA from the positive clones for sequencing. Use forward and reverse primers basedon the DNA vector used for cloning. It is recommended to submit 10 colonies for sequencing to ensure representative results. It is also necessary to submit unconverted DNA for
sequencing comparison.

Step 8: Analyzing the sequencing results
1. If the CpG dinucleotide was methylated in the original DNA, the sequencing read will show
a CG at that location. If the CpG dinucleotide was unmethylated in the original DNA, thebisulfite conversion will have converted the cytosine to uracil and the sequencing read willshow a TG at that location.
2.

Compare the bisulfite converted sequence data against the unconverted DNA sequence.

3.

Quantify the number of methylation sites for the region of interest to determine the percentage of methylation.

www.activemotif.com

12

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.

Frommer, M. et al. (1992) PNAS, 89: 1827.
Clark, S.J. et al. (1994) Nuc. Acids Res., 22: 2990-2997.
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.
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.
Fang, J.Y. et al. (2004) Chin Med J (Eng.), 117(1): 99-103.
Li, LC and Dahiya R. (2002) Bioinformatics, 18(11): 1427-31
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.

www.activemotif.com

13

Appendix
Section A. Troubleshooting Guide
Problem/question

Recommendation

Using less than 100 ng of
DNA as starting material

When converting a small amount of DNA, include 250 ng Salmon Sperm DNA as a carrier
DNA in the conversion reaction to minimize loss. Use nested PCR primers if possible.

Starting DNA volume isgreater than 13 µl

DNA volumes up to 20 µl can be used without affecting the protocol

Conversion time

A 5 hour incubation at 50°C is normally sufficient to efficiently convert DNA samples.
However, DNA sequences that are GC-rich and/or contain extensive secondary structures
may require a 9 hour conversion at 50°C. If noticing a loss of DNA after the conversion
reaction, the conversion time can be reduced to as little as 3 hours.

Can I store my conversion The DNA is recommended to be stored at 4°C for 5 days. If longer storage is needed, store
reaction samples forsamples at -20°C. As precipitates can form during -20°C, long term storage is not recomlonger than 5 days before
mended.
performing the DNA
purification and desulfonation steps?
Storage of converted DNA

Once DNA is prepared using MethylDetector, samples may be stored at -20°C prior to PCR
analysis. However, we do 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.

Section B. Related Products
DNA Methylation		
MethylCollector™		
MethylCollector™ Ultra		
UnMethylCollector™		
Fully Methylated Jurkat DNA		

Format25 rxns
30 rxns
30 rxns
10 µg

Catalog No.
55002
55005
55004
55003

Antibodies

Application

FormatCatalog No.

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

100 µl50 µg
200 µl100 µg100 µg100 µg100 µg200 µg100 µg200 µl100 µg100 µg100 µg100 µg200 µ1

39769
39649
39203
39204
39205
39206
39207
39365
39215
39547
39216
39217
39218
39741
39625

Chromatin Immunoprecipitation		

FormatCatalog No.

ChIP-IT™ Express		ChIP-IT™ Express Enzymatic		

25 rxns
25 rxns

53008
53009

5-Hydroxymethylcytidine pAb
5-Methylcytidine mAb
CGBP rabbit pAb
DNMT1 mouse mAb
DNMT2 rabbit pAb
DNMT3A mouse mAb
DNMT3B mouse mAb
Kaiso mouse mAb
MBD1 mouse mAb
MBD2 rabbit pAb
MBD3 mouse mAb
MBD4 rabbit pAb
MeCP2 rabbit pAb
Ubiquitin mouse mAb
Uhrf1 rabbit pAb

www.activemotif.com

14

ChIP-IT™ Express HT		
Re-ChIP-IT™		
ChIP-IT™ Protein G Magnetic Beads		
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		Bridging Antibody for Mouse IgG		

96 rxns
25 rxns
25 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns
500 µg

53018
53016
53014
53010
53011
53012
53015
53019
53020
53021
53017

Recombinant Methylated Histones		

FormatCatalog No.

Recombinant Histone H2A		
Recombinant Histone H2B		
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
50 µg

31251
31252
31207
31208
31209
31210
31211
31212
31213
31214
31215
31216
31217
31218
31219
31220
31221
31222
31223
31224
31225
31226

Histone Purification & Chromatin Assembly		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		
Chromatin Assembly Kit		
HeLa Core Histones		

10 rxns
10 rxns
10 rxns
36 µg

40025
40026
53500
53501

Histone Acetyltransferase and Deacetylase Activity

FormatCatalog No.

HAT Assay Kit (Fluorescent)		
Recombinant p300 protein, catalytic domain		
Recombinant GCN5 protein, active		
HDAC Assay Kit (Fluorescent)		
HDAC Assay Kit (Colorimetric)		

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

56100
31205
31204
56200
56210

Co-Immunoprecipitation		

FormatCatalog No.

Universal Magnetic Co-IP Kit		
Nuclear Complex Co-IP Kit		

25 rxns
50 rxns

54002
54001

www.activemotif.com

15

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
104 Avenue Franklin Roosevelt
B-1330 Rixensart, 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

16