Hydroxymethyl Collector™

Catalog No. 55013(version B2)

Active Motif North America
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Copyright 2013 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.
© 2013 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  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 2Introduction  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5Hydroxymethyl Collector™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Sample Preparation: Fragmentation of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
Hydroxymethyl Collector™ Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Appendix
Section A. Use of Included 5-hmC Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Section B. Use of Magnetic Beads and Included Magnet . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section C. PCR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Section D. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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Overview
The Hydroxymethyl Collector™ Kit is designed to detect and capture DNA fragments containing
5-hydroxymethylcytosine (5-hmc) DNA methylation*. The kit works with double-stranded DNA
that has been fragmented to a size range of 100-500 base pairs. The DNA is then incubated in
the presence of a b-glucosyltransferase enzyme and a modified UDP-glucose donor. The enzyme
transfers the glucose to 5-hydroxymethylcytosine residues, creating glucosyl-hydroxymethylcytosine. A biotin conjugate is then chemically attached to the modified glucose. Magnetic streptavidin beads and the included bar magnet are used to capture the biotinylated 5-hmC DNA fragments.
The elution buffer releases the DNA fragments from the biotin conjugation, leaving you with
DNA that is enriched in 5-hmC methylation. Following purification, with the included purification
reagents, the enriched DNA can be used for analysis of individual genes by PCR, or in combination
with microarrays and sequencing for genome-wide 5-hydroxymethylcytosine analysis.
By utilizing chemical labeling of 5-hmC residues, the Hydroxymethyl Collector Kit is extremely
specific in its capture of hydroxymethylated DNA fragments. The biotin-streptavidin binding reaction also allows for more stringent binding and wash conditions, enabling enrichment of 5-hmC
residues that cannot be detected by antibody immunoprecipitation methods. Active Motif’s fast,magnetic protocol has been streamlined to minimize the number of wash and incubation steps,
delivering you enriched 5-hmC DNA in less than 4 hours. For added convenience, the kit also
includes a 5-hydroxymethylcytosine DNA control and PCR primers that can be used to verify theefficiency of the enrichment.

product

formatcatalog no.

Hydroxymethyl Collector™

25 rxns

55013* Patent Pending
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1Flow Chart of Process
Fragmented DNA
containing 5-hmC

β-Glucosyltransferase enzyme+ UDP-Azide-Glucose
1 hour at 37˚C

N3

Modified Glucosyl-5-hmC

BiotinBiotin Conjugate Solution
1 hour at 37˚C

N3

Biotinylated Glucosyl-5-hmC

SA
BiotinMagnetic Streptavidin Beads
30 minutes at RT

N3

DNA is captured for washing and elution

SA
BiotinElution of DNA from the Biotin
30 minutes at RT

N3

Eluted DNA is purified using theincluded purification reagents

Enriched DNA containing
5-hydroxymethylcytosine

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2

Introduction
5-Hydroxymethylcytosine DNA Methylation (5-hmC)
In mammals and other vertebrates, DNA methylation usually occurs at the C5 position of cytosine(5-mC), mostly within CpG dinucleotides. In 2009, Kriaucionis and Heintz and Tahiliani et al. discovered another DNA modification, 5-hydroxymethylcytosine (5-hmC), which was observed to beelevated in neurons and embryonic stem cells1,2. The 5-hydroxymethylcytosine modification resultsfrom the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET
family of cytosine oxygenases2,3. While the precise function of 5-hmC has yet to be determined, it
has been postulated that it could represent a pathway to demethylate DNA, as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine3. Alternatively,
5-hmC may be produced by the addition of formaldehyde to DNA cytosines by DNMT proteins4.
One of the difficulties in studying 5-hmC is the fact that many of the traditional techniques
employed to study DNA methylation, such as bisulfite sequencing or certain methylation-sensitive
restriction enzymes, do not distinguish between 5-mC and 5-hmC residues3,5,6. Methyl CpG binding
protein enrichment methods only serve to selectively bind 5-mC DNA methylation, but cannot be
used to enrich for 5-hmC5.
In order to overcome some of the difficulties with direct discrimination of 5-mC and 5-hmC
residues, a T4 bacteriophage enzyme, b-glucosyltransferase, has been used to modify 5-hydroxymethylcytosine residues. The b-glucosyltransferase enzyme utilizes a UDP-glucose (uridine
diphosphoglucose) donor to attach a glucose moiety to 5-hmC which generates glucosyl-hydroxymethylcytosine7. With the sugar attached to 5-hmC, the two types of DNA methylation canbe discriminated using glucosyl-sensitive restriction enzymes.
Another method used to discriminate between 5-mC and 5-hmC is using antibodies specificfor each form of DNA methylation. These antibodies can be used to detect or enrich for DNA
fragments containing the methylation of interest. This antibody immunocapture technique isknown as Methylated DNA Immunoprecipitation (MeDIP)8. One of the drawbacks of the antibody
immunoprecipitation method is the enrichment of DNA fragments is limited by the quality of theantibody and the stringency of the binding conditions. High stringency conditions may eliminate
weak binding of 5-hmC, while low stringency conditions may increase non-specific binding.
Active Motif’s Hydroxymethyl Collector Kit overcomes some of the difficulties with antibody
enrichment methods, while still retaining the ability to clearly discriminate between 5-mC and5-hmC residues. The Kit utilizes the modification properties of b-glucosyltransferase to specificallyalter 5-hmC with a modified glucose. A biotin conjugate is chemically introduced and streptavidin
magnetic beads are used to enrich for DNA fragments containing the modified 5-hydroxymethylcytosine fragments9. The strong binding properties of biotin-streptavidin enable high stringency
washes without sacrificing the sensitivity of the assay. Eluted DNA is purified and ready to use indownstream analysis by PCR, microarray or sequencing.

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3

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 or 5-hydroxymethylcytosine can be used to determine the methylation status of specific CpG dinucleotides10.
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 conversion consists of the treatment of genomic DNA withsodium bisulfite, leading to deamination of unmethylated cytosines into uracil. PCR isthen performed with primers that differentiate between methylated and unmethylatedsequences11. Bisulfite-based techniques can be cumbersome, involving time- and laborintensive chemical treatments that damage DNA and limit throughput. Additionally, bisulfite
conversion doesn’t differentiate between 5-methylcytosine and 5-hydroxymethylcytosine3,5,6.
3. Methyl-CpG Binding proteins: This family of proteins takes its definition from themethyl-CpG binding domain (MBD), the minimum portion with specific affinity for a single,
symmetrically methylated CpG pair. 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 DNA12. The Methylated CpG Island
Recovery Assay (MIRA) utilizes the combination of MBD2b with its binding partner MBD3L1,
methyl-CpG-binding protein 3-like-1, to generate a higher affinity for methylated DNA than
MBD2b protein alone13, 14. Methyl-CpG binding proteins are limited to the evaluation of
methylated DNA in a CpG context as the proteins do not recognize methylated cytosinesthat exist outside of a CpG dinucleotide. The MBD proteins are only capable of binding to
5-mC methylation; they cannot be used to enrich for 5-hmC methylation5.
4. Methylated DNA Immunoprecipitation (MeDIP): Methylated DNA Immunoprecipitation
is a an immunocapture technique in which an antibody specific for methylated cytosines
is used to immunoprecipitate methylated genomic DNA fragments8. The affinity of theantibody used in MeDIP enables the detection of methylated cytosines regardless of theircontext. This means that MeDIP can be used for the detection of any methylated cytosine
and is not restricted to analysis of CpG methylation. The enriched DNA can be used forindividual analysis of the methylation status of a particular gene by PCR, or in combination
with microarrays for genome-wide methylation analysis. MeDIP can also be used to prepare
samples for use in high-throughput sequencing (HTS) techniques. Antibodies are available forthe specific detection of either 5-methylcytosine or 5-hydroxymethylcytosine.

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4Kit Performance and Benefits
The Hydroxymethyl Collector kit is designed to specifically enrich for DNA fragments containing
5-hydroxymethylcytosine (5-hmC).
Range of detection: We recommend using between 5 ng and 2.5 µg fragmented genomic DNA
per capture reaction for detection by qPCR. Larger quantities of DNA are not efficiently captured(see image below). For downstream analysis by hybridization to a microarray, it may be necessary
to pool multiple enrichment reactions or perform whole-genome amplification following 5-hmC
DNA enrichment.
For genome-wide analysis by deep sequencing, larger amounts of DNA may be needed. To date
Active Motif has not validated the required amount of DNA needed to perform deep sequencing.
Suggested input DNA amounts are 8-10 µg from stem cells, 20 µg from cancer cells and 1-3 µg frombrain, but optimization may be necessary. To acquire enough sample material for deep sequencing,
we recommend performing multiple enrichment reactions and pooling the DNA together.
Cross-reactivity: The b-glucosyltransferase reaction is specific to 5-hydroxymethylcytosine andwill not react with unmethylated or 5-methylcytosine residues. The chemical conjugation of biotinis specific to the modified glucose moiety included in the kit and will not react with unmodified
glucose. This enables precise capture of 5-hydroxymethylcytosine. Hydroxymethyl Collector issuitable with any species (human, mouse, rat, etc.) containing 5-hydroxymethylcytosine residues.
Assay time: 4 hours

Hydroxymethyl Collector™
Hydroxymethyl Collector range of detection.

Hydroxymethyl Collector™

1210% of Input

8
6
4
2
0

5 ng

25 ng 100 ng 500 ng 2.5 µg
Mouse brain DNA

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5 µg

10 µg

Mouse brain DNA was assayed at various quantities
in the Hydroxymethyl Collector Kit. Enriched DNA
was analyzed by qPCR across multiple loci. The enKcnab1
riched DNA was plotted as a percentage of starting
Slc22a4
material. This graph shows that Hydroxymethyl
Faim2Control 2 Collector has a preferred range of detection from
5 ng to 2.5 µg for mouse brain DNA. Larger DNA
quantities are not efficiently captured. The
Control 2 primer set serves as a negative control inthis experiment.

5

% of Input

18
16
14
12
10
8
6
4
2
0

Hydroxymethyl Collector™
5-hmC
No glucose

Sample 1

Sample 2

Hydroxymethyl Collector qPCR results using human genomic DNA and “spiked” 5-hmC control.
MseI digested human genomic DNA (500 ng) was“spiked” with 25 pg of the included 5-hmC Control
DNA in duplicate. These samples were thenprocessed using the Hydroxymethyl Collector Kit
to enrich for 5-hydroxymethylcytosine residues
in either the presence (5-hmC) or absence (Noglucose) of the UDP-Azide-Glucose donor. Eluted
DNA was purified and tested in real time PCR using
the included APC PCR primer mix to screen for
the enrichment of the 5-hmC Control DNA. The
DNA was quantified against the Input standard
curve and plotted as a percentage of the starting
material. This graph shows that Hydroxymethyl
Collector is able to enrich for 5-hmC DNA and thatthe enrichment only occurs in the presence of the
UDP-Azide-Glucose donor.

Hydroxymethyl Collector Kit Components and Storage
Hydroxymethyl Collector Kits are for research use only. Not for use in diagnostic procedures. Allcomponents are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storageb-glucosyltransferase enzyme (200 U/µl)

50 µl

-20°C

UDP-Azide-Glucose (3 mM)

65 µl

-20°C

10X Reaction Buffer AM1

500 µl-20°C

1 M DTT

100 µl-20°C

Biotin Conjugate Solution

500 µl-20°C

Streptavidin Beads*

700 µl4°C

Binding Buffer AM13

50 ml

4°C

10X Elution Buffer AM2

250 µl4°C5-hmC Control DNA (50 ng/µl)

10 µl

-20°C

Human genomic DNA, MseI digested (20 ng/µl)

300 µl-20°C

APC PCR Primer Mix (2.5 µM)

400 µl-20°C

DNA Purification Binding Buffer

50 ml

RT

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6

DNA Purification Wash Buffer**

50 ml

RT

DNA Purification Elution Buffer

5 ml

RT

3M Sodium Acetate***

500 µlRT

DNA purification columns

50 ea

RT

DNA column collection tubes

50 ea

RT

0.2 ml PCR tubes

1 bag

RT

Bar magnet and glue dots

1 ea

RT

* Do not freeze the Streptavidin Beads. Upon receipt of this kit, store beads at 4°C.
** DNA Purification Wash Buffer must be reconstituted to a final concentration of 80% ethanol
prior to use.
*** Do not store the 3M Sodium Acetate at 4°C.
Additional materials required• Sample DNA that has been fragmented between 100-500 bp in size
• End-to-end rotator (e.g. Labquake from Barnstead/Thermolyne)• Ethanol, 100%
• 1.5 or 2 ml microcentrifuge tubes• Microcentrifuge• DNase-free sterile waterwww.activemotif.com

7

Protocols
Buffer Preparation and Recommendations
Samples
Hydroxymethyl Collector reactions can be performed using 5 ng - 2.5 µg of fragmented genomic
DNA. Fragments should range in size from 100-500 bp. Use the recommended protocols for
preparing fragmented DNA by restriction enzyme digestion or sonication prior to starting theHydroxymethyl Collector assay.
Set aside a quantity of fragmented genomic DNA equivalent to 10% of the DNA being used in thecapture reactions for use as Input DNA (e.g. for 500 ng DNA in capture reaction, set aside 50 ng
DNA for Input). Separate Input DNA should be saved for each DNA source tested. The Input DNA
can be used in real time PCR to quantify the amount of enriched DNA recovered from the biotin
capture reaction. For details on real time PCR analysis and the use of Input DNA, see Appendix C.
Store the Input DNAs at -20°C until ready to use.
5-hmC Control DNA
A 338 base pair fragment containing 122 cytosine residues from the APC (adenomatosis polyposiscoli) gene promoter was amplified by PCR. The control DNA contains 25% 5-hydroxymethylcytosine residues instead of traditional cytosines.
A separate control reaction can be set up to verify the efficiency of 5-hydroxymethylcytosine
DNA capture. For details on setting up a positive control reaction, please refer to Appendix A on
page 17. The 5-hmC Control DNA should be “spiked” into sample DNA, or the included humangenomic DNA, according to the protocol and the capture reactions performed as indicated. We
recommend maintaining a control DNA spike to fragmented DNA ratio of 1:20,000 (e.g. 25 pg spike
DNA standards in 500 ng fragmented genomic DNA = 1:20,000 ratio). The control DNA is provided
at a concentration of 50 ng/µl.
Human genomic DNA, MseI digested
Human genomic DNA that has been digested with MseI is provided as a control for the kit. The
5-hmC Control DNA can be “spiked” into the human genomic DNA to verify the efficiency of theenrichment reactions. Please refer to Appendix A for details on setting up the positive control
reaction. The Human genomic DNA is provided at a concentration of 20 ng/µl.
10X Reaction Buffer AM1
The reaction buffer is provided ready to use at a 10X concentration. Please follow the instructionsin the protocol.
1M DTT
Prepare a fresh 10 mM working stock of DTT by diluting 5 µl 1M DTT into 495 µl sterile water eachtime reactions are set up. Vortex to mix. Avoid multiple freeze thaws of DTT as this will diminish
its effectiveness. Discard any unused 10 mM DTT.

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8

b-Glucosyltransferase Enzyme
Is provided ready to use. The enzyme is supplied at 200 U/µl concentration.
UDP-Azide-Glucose
The modified glucose moiety is provided at a 3 mM stock concentration and is ready to use.
Biotin Conjugate Solution
Is provided ready to use.
DNA Purification Wash Buffer
The DNA Purification Wash Buffer requires the addition of ethanol before use. The final concentration of ethanol should be 80%. Add 40 ml of fresh 100% ethanol to the DNA Purification Wash
Buffer bottle. Invert repeatedly. The buffer can be stored at room temperature after the additionof ethanol. The ethanol only needs to be added before the first use, after that the Wash Buffer isready for use.
3M Sodium Acetate
It is important to check the sodium acetate before use to ensure that the salts have not precipitated out of solution. Once the sodium acetate is in solution it should be stored at room
temperature.
Binding Buffer AM13
Is provided ready to use.
10X Elution Buffer AM2
Prepare a 1X Elution Buffer by diluting the 10X Elution Buffer AM2 in Binding Buffer AM13. For every
100 µl elution, dilute 10µl 10X Elution Buffer AM2 into 90 µl Binding Buffer AM13.

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9Assay Protocol
Read the entire protocol before use.
NOTES BEFORE STARTING
Sample Preparation: Fragmentation of Genomic DNA
Prior to starting the Hydroxymethyl Collector assay, genomic DNA should be fragmented using
either mechanical fragmentation (e.g. sonication) or restriction digestion with a methylationinsensitive restriction enzyme to yield fragments ranging in size from 100 to 500 base pairs.
Hydroxymethyl Collector will enrich for 5-hydroxymethylcytosines. If trying to evaluate methylation of CpG dinucleotides, such as in CpG islands, please follow the recommendations below forfragmentation of genomic DNA.
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 thisregion (see “Appendix D. Troubleshooting” for further discussion).
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.

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10

Example Fragmentation Protocols
Example fragmentation protocols are provided for both restriction digest and mechanical fragmentation. We suggest using 4 µg of purified genomic DNA when performing restriction digestionand 20 µg of purified genomic DNA for sonication. Hydroxymethyl Collector reactions can be
performed on 5 ng - 2.5 µg of fragmented genomic DNA.
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		

Note 1:

10 µl
10 µl
1 µl
1 µl
78 µl
100 µlThe DNA volume may vary depending on its initial concentration.

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
by phenol/chloroform extraction and precipitation, or through use of a DNA purification
column (such as Active Motif’s Chromatin IP DNA Purification Kit, Catalog No. 58002).
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.
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), with a 20-second pause on ice between eachpulse. The sheared DNA can be visualized by ethidium staining after electrophoresis on a 3%
agarose gel.
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11

Hydroxymethyl Collector Protocol
Step A: Glucosylation Reaction1.

Set up a 200 µl PCR tube for each glucosylation reaction to be performed. If desired, a control reaction should be set up with the included 5-hydroxymethylcytosine DNA. For notes onusing the included control DNA, please see Appendix A on page 17.

2.

Calculate the amount of reagent needed for each capture reaction.
Fragmented DNA: Recommended range between 5 ng and 2.5 µg. Set aside a quantity offragmented genomic DNA equivalent to 10% of the DNA being used in the glucosylation
reaction for use as Input DNA and store at -20°C. Separate Input DNA should be saved foreach DNA source.
Sterile water: Determine the amount of sterile water needed to bring the reaction to a
final volume of 50 µl.

3.

Add reagents in the order listed below to each PCR tube.

Reagents

SampleNegative
Control

Sterile water______ µl______ µl

10X Reaction Buffer AM1

5 µl

5 µl

10 mM DTT

5 µl

5 µl

UDP-Azide-Glucose

2.5 µl–Fragmented DNA______ µl______ µlb-Glucosyltransferase (200 U/µl)

2 µl

2 µl

Total Volume

50 µl

50 µl

4.

Cap the PCR tubes tightly. Vortex the reaction to ensure complete mixture. Quick spin the
tubes to collect the material to the bottom.

5.

Incubate the reaction at 37°C for 1 hour.

Step B: Biotinylation Reaction
1.

Quickly spin the PCR tubes to collect the contents at the bottom.

2.

Add 20 µl Biotin Conjugation Solution to each reaction.

3.

Cap the PCR tubes tightly. Vortex the reaction to ensure complete mixture. Quick spin the
tubes to collect the material to the bottom.

4.

Incubate the reaction for 1 hour at 37°C.

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12

Step C: Purification of Biotinylated DNA
1.

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

2.

To each biotinylated DNA reaction, add 5 volumes DNA Purification Binding Buffer for every
volume of DNA sample. For example, if your reaction is 70 µl, add 350 µl DNA Purification
Binding Buffer.

3.

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

4.

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

5.

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

6.

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

7.

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

8.

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

9.

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

10. Transfer the column to a clean microcentrifuge tube.
11.

Add 50 µl of DNA Purification Elution Buffer to the center of the column matrix and wait for
1 minute.
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13

12.

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

13.

Discard column and proceed to Step D below.

Step D: Capture Reaction
1.

Resuspend the Streptavidin Beads by vortexing or flicking the tube. Make sure the beads are
fully resuspended before use. If preparing multiple reactions, ensure beads do not settle byoccasional pipetting to resuspend the beads before addition to the capture reactions.

2.

In fresh 0.2 ml PCR tubes prepare the following reactions:

3.

(Optional)
Positive
Control

Reagents

SampleNegative
Control

Streptavidin Beads

25 µl

25 µl

25 µl

Binding Buffer AM13

25 µl

25 µl

25 µl

Purified Biotinylation reaction

50 µl

50 µl

50 µl

Total Volume

100 µl100 µl100 µlIncubate the reaction for 1 hour at room temperature with end-to-end rotation.

Step E: Washing and Elution
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, place supernatant in a microcentrifuge tube and store at -20°C. Otherwise,
carefully remove and discard the supernatant. To use the magnet provided in the kit, please
see Appendix B.

2.

Wash beads five times with 200 µl Binding Buffer AM13. Pipette 2-3 times gently to resuspend.
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 AM13 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.

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.

4.

Resuspend the washed beads with 100 µl 1X Elution Buffer AM2 by pipetting 2-3 times.
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14

5.

Incubate for 30 minutes at room temperature with end-to-end rotation.

6.

Briefly centrifuge the tubes to collect liquid from the cap.

8.

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

9.

Transfer the supernatant, which contains the enriched DNA, to a fresh tube.

Step F: Purification of Enriched DNA
1.

To each enriched DNA reaction, add 5 volumes DNA Purification Binding Buffer for every
volume of DNA sample. For example, if your reaction is 100 µl, add 500 µl DNA Purification
Binding Buffer.

3.

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

4.

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

5.

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

6.

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

7.

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

8.

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

9.

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

10. Transfer the column to a clean microcentrifuge tube.
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15

11.

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

12.

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

13.

Discard column. The DNA eluted into the microcentrifuge tube is purified and ready to use inyour downstream application.

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

Kriaucionis, S. and Heintz, N. (2009) Science, 324: 924-929.
Tahiliani, M. et al. (2009) Science, 324: 930-935.
Huang, Y. et al. (2010) PLoS One, 26: e8888.
Liutkeviciute, Z. et al. (2009) Nat. Chem. Biol., 5: 400-402.
Jin, S. et al. (2010) Nucl. Acids Res., 38: e125.
Nestor, C. et al. (2010) BioTechniques, 48: 317-319.
Szwagierczak, A. et al. (2010) Nucl. Acids Res., 38: e181.
Weber, M. et al. (2005) Nat. Genet., 37: 853-832.
Song, C. et al. (2010) Nat. Biotechnology, 29: 68-72.
Dalh, C. and Guldberg, P. (2003) Biogerontology, 4: 233-250.
Frommer, M. et al. (1992) Proc. Natl Acad. Sci. USA, 89: 1827-1831.
Fraga, M.F. et al. (2003) Nucleic Acids Res., 31: 1765-1774.
Rauch, T. and Pfeifer, G. (2005) Lab. Investigation, 85: 1172-1180.
Jiang, C.L. et al. (2004) J. Bio. Chem., 279: 52456-52464.

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16

Appendix
Section A. Use of Included 5-hMC Control DNA
Control 5-hydroxymethylated DNA is included as an optional validation of the enrichment
efficiency. When control 5-hydroxymethylcytosione DNA is “spiked” into genomic DNA, it will
undergo the glucosylation and biotinylation reactions and will be captured by the streptavidin
beads. Following elution and purification, the DNA can be analyzed in real time PCR using theprovided APC PCR primer mix to verify that the efficiency of the capture method. The APC locus
in not methylated in normal, human genomic DNA, therefore if the source of the genomic DNA
used is normal human, the only enrichment you should expect is from the “spiked” 5-hmC DNA.
MseI digested human genomic DNA is provided for use as a control in setting up the spiked reaction. Alternatively, sample genomic DNA can be used to set up the spiked reactions. For sourcesof genomic DNA other than normal human, you will need to run a background reaction containing
only the sample DNA, without any control DNA, alongside the spiked reactions to determine theamount of enrichment from naturally occurring APC methylation in your genomic DNA sample
versus the amount of enrichment due to the control DNA spike. There should be an increase inenrichment in spiked samples as compared to genomic DNA alone.
1.

Set up a 200 µl PCR tube for each glucosylation reaction to be performed.

2.

Calculate the amount of reagent needed for each glucosylation reaction.
Fragmented DNA: Recommended range between 5 ng and 2.5 µg. Set aside a quantity offragmented genomic DNA equivalent to 10% of the DNA being used in the capture reaction
for use as Input DNA and store at -20°C. Separate Input DNA should be saved for each DNA
source.
Sterile water: Determine the amount of sterile water needed to bring the glucosylation
reaction to a final volume of 50 µl.
Control 5-hmC DNA: The amount of spiked control 5-hmC DNA should be determinedbased on the amount of DNA used in the capture reaction. We recommend maintaining a
spiked control DNA to fragmented DNA ratio of 1:20,000.
Prepare a 10% excess of spiked control 5-hmC DNA in fragmented genomic DNA. Mix well byvortexing. Remove 10% of the spiked genomic DNA reaction and set aside for use as Input
DNA for quantification during real time PCR. Input DNA can be stored at -20°C until ready touse. Some example calculations are shown below for reference.

Control
spike DNA

Fragmented
Quantity Final control
Genomic removed for spike DNA
DNA
Input DNAper rxn

Final genomic DNA
per rxn

Ratio ofspike togenomic

5.5 pg110 ng10% volume

5 pg

100 ng

1:20,000

27.5 pg

550 ng10% volume

25 pg

500 ng

1:20,00055 pg

1.1 µg10% volume

50 pg

1 µg

1:20,000www.activemotif.com

17

5-hmC Control DNA is provided at a concentration of 50 ng/µl. Please follow the recommendations in
the table below for preparing the “spike” control DNA into the provided human genomic DNA or yoursample genomic DNA. Example calculations are shown below for different genomic DNA concentrations. The included Human genomic DNA is provided at a concentration of 20 ng/µl. We recommend
performing the first dilution of control DNA in sterile water (Tube 1), followed by subsequent dilutions
into genomic DNA at the appropriate stock concentration. The dilutions are performed in genomic
DNA in order to avoid over-diluting the genomic DNA concentration used in the final capture reactions.
Use the control DNA at the provided concentration. Mix each dilution step by vortexing.
Sample genomic
DNA concentration

4 ng/µl

20 ng/µl

40 ng/µl

Spike + genomic
DNA quantity

5 pg in 100 ng

25 pg in 500 ng

50 pg in 1 µg

Tube 1

5 µl control DNA into
5 ml H20 = 50 pg/µl

5 µl control DNA into 5 µl control DNA into
995 µl H20 = 0.25 ng/µl 495 µl H20 = 0.5 ng/µl

Tube 22 µl Tube 1 into
18 µl genomic DNA= 5 pg/µl

2 µl Tube 1 into
18 µl genomic DNA= 25 pg/µl

2 µl Tube 1 into
18 µl genomic DNA= 50 pg/µl

Tube 32 µl Tube 2 into
48 µl genomic DNA= 0.2 pg/µl

2 µl Tube 2 into
48 µl genomic DNA= 1 pg/µl

2 µl Tube 2 into
48 µl genomic DNA= 2 pg/µl

Input DNA volume

2.5 µl Tube 3

2.5 µl Tube 3

2.5 µl Tube 3

Reaction volume

25 µl Tube 3

25 µl Tube 3

25 µl Tube 3

3.

Set up glucosylation reactions by adding reagents in the order listed to each PCR tube.
Sample and Negative control reactions should just include fragmented DNA, while the Positive control sample will use the spiked DNA prepared in Step 2 above.
SpikedPositive
Control

Reagents

SampleNegative
Control

10X Reaction Buffer AM1

5 µl

5 µl

5 µl

10 mM DTT

5 µl

5 µl

5 µl

b-Glucosyltransferase (200 U/µl)

2 µl

2 µl

2 µl

UDP-Azide-Glucose

2.5 µl–

2.5 µlFragmented DNA______ µl______ µl

25 µl

Sterile water______ µl______ µl10.5 µl

Total Volume

50 µl

50 µl

50 µl

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18

4.

Cap the PCR tubes tightly. Vortex the reaction to ensure complete mixture. Quick spin the
tubes to collect the material to the bottom.

5.

Incubate the reaction at 37°C for 1 hours with end-to-end rotation.

6.

Proceed with Step B: Biotinylation Reaction on page 12.

Notes about Input DNA• Sample IP Input: To be used with the PCR primers designed to analyze the sample DNA.
This standard curve will be used to determine the amount of enriched 5-hydroxymethylcytosine DNA present in the final elution. This Input can be used to quantify DNA in the
Sample IP reactions and the Negative control reactions.
• 5-hmC DNA spike Input: To be used with the provided APC PCR primer set. This standard
curve will be used to determine the amount of 5-hmC DNA captured in the final elution of
the positive control reaction.

www.activemotif.com

19Section B. 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 Binding Buffer AM13
in one tube of an 8-well strip of PCR tubes. Use this tube with the assembled bar
magnet stand to become familiar with use of the beads and magnet. It is difficult

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20

to re-suspend the beads if the tubes are directly adjacent to the magnet, so it isusually 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 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.

www.activemotif.com

21Section C. PCR Analysis
PCR Primer Design
The Hydroxymethyl Collector Kit includes an APC PCR primer mix for use with the provided control DNA. If possible, real time PCR is recommended for analysis of DNA enriched by Hydroxymethyl Collector. 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 end point PCR or an amplicon of 100-150 bp for real time PCR.
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 the 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. Primers that dimerize should be avoided as they
compromise accurate quantitation.

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 24. Use the slope of theplotted standard curve in the primer efficiency equation above.

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22Real Time PCR Analysis
It is important to use purified DNA prior to use in downstream applications. The samples andInput DNAs should be subjected to a DNA clean-up step prior to real time PCR analysis. Below is
an example PCR reaction. Please follow the specific instructions for your real time PCR instrument.
1.

For notes on preparing standard curves with the Input DNA, please see page 24.

2.

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 APC PCR Primer Mixes contain both Forward and Reverse primers for use
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.
Note:
3.

Place tubes in a real time PCR instrument and program as below. The amplification conditions should be optimized for each target locus, master mix reagent and PCR instrument. A
suggested starting point is:		4.

It is recommended to prepare triplicates of each sample and Input reaction.

95°C for 2 minutes(95°C for 3 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.

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23

Data Analysis and Use of Input DNAs
5-Hydroxymethylcytosine DNA isolated using Hydroxymethyl Collector can be analyzed by PCR
amplification of the loci of interest. However, if the goal is to compare the methylation status ofparticular loci in different DNA samples, it is essential that Hydroxymethyl Collector be performedon the same amount of each DNA sample. In addition, Input DNA should be prepared for each of
the different DNA samples 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.

For Hydroxymethyl Collector DNA, produce a standard curve at 3.3, 0.33, 0.033 and 0.0033
ng/µl in triplicate using the Input DNAs captured in Step A instruction #2. We recommend
running a standard curve every time the PCR amplification is performed. An example standard curve calculation is provided on page 26.

2.

Run each sample with the appropriate Input DNA standard (i.e. prepare a separate standard
curve for each DNA source or spiked control tested.
• Sample IP Input: To be used with the PCR primers designed to analyze the sample DNA.
This standard curve will be used to determine the amount of enriched 5-hydroxymethylcytosine DNA present in the final elution. This Input can be used to quantify DNA in both the
Sample IP reactions and the Negative control reactions.
• (Optional) 5-hmC DNA spike Input: To be used with the provided APC PCR primer set. This
standard curve will be used to determine the amount of 5-hmC DNA captured in the final
elution of the Positive control reaction.

3.

Every gene and/or primer set will generate a different amplification profile.

4.

CT = Threshold Cycle and is the cycle number where the signal exceeds the backgroundthreshold level. CT values should be plotted for each gene to create a linear regression plot.

5.

Plot CT versus log DNA concentration. See Figure 1 on page 25. The slope of the standard
curve can be used to determine primer efficiency in the equation on page 22.

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24

35

30

y = -1.8593Ln(x) + 19.918

CT

R2 = 0.9856

25

20

15
0.001

0.01

0.1

1

10

100

DNA concentration (ng/µl)

Figure 1: Example standard curve linear regression plot.
A standard curve for Input DNA is provided as a reference only. Ten-fold dilutions of Input DNA were tested in triplicate
and plotted against the CT value. A new standard curve should be generated each time the assay is performed.

6.

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

7.

Calculate the percent enrichment. Use the sample DNA quantity calculated above andcompare it with the amount of DNA used in the initial glucosylation reaction (Step A).

		% of Input =		Amount of enriched Sample DNA material (ng)Amount of fragmented DNA used in the initial reaction (ng)		Fold enrichment =		

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Amount of enriched Sample DNA material (ng)Amount of enriched Negative control material (ng)25x 100%Example Standard Curve Calculations
To illustrate how to set up a standard curve at the recommended concentrations of 3.3,
0.33, 0.033 and 0.0033 ng/µl, example calculations are shown below for the preparation ofa standard curve using Input DNA from the optional spike reaction containing 25 pg spike
DNA in 500 ng genomic DNA. It is critical to adjust DNA concentrations and DNA quantities
based on the actual amount of spike DNA and genomic DNA used in the reactions in orderto obtain accurate quantification of eluted material. Perform the following serial dilutions togenerate the DNA standards listed in the table below.
Input: Contains 10% of initial reaction quantity. (2.5 pg spike DNA in 50 ng genomic DNA)
Tube 1: Take the 2.5 µl Input DNA and add 12.5 µl sterile water. Vortex to mix.
Tube 2: Make a 1:10 dilution by transferring 5 µl DNA from Tube 1 into 45 µl sterile water.
Vortex to mix.
Tube 3: Make a 1:10 dilution by transferring 5 µl DNA from Tube 2 into 45 µl sterile water.
Vortex to mix.
Tube 4: Make a 1:10 dilution by transferring 5 µl DNA from Tube 3 into 45 µl sterile water.
Vortex to mix.

Tube #

Genomic
DNA conc.

Spike
DNA conc.

Final
volumeVolumeper PCR

Genomic
DNA qty.

Spike
DNA qty.

Input

50 ng

2.5 pg2.5 µl

N/A

N/A

N/A

1

3.3 ng/µl

0.166 pg/µl

15 µl

3 µl

10 ng/well

0.5 pg/well20.33 ng/µl

0.0166 pg/µl

50 µl

3 µl

1 ng/well

0.05 pg/well

30.033 ng/µl0.00166 pg/µl

50 µl

3 µl

0.1 ng/well0.005 pg/well

40.0033 ng/µl 0.000166 pg/µl

50 µl

3 µl

0.01 ng/well

0.0005 pg/well

Use 3 µl of DNA from Tubes 1-4 to set up PCR reactions for the standard curve.
Plot the CT vs. log genomic DNA concentration as shown on page 25.

www.activemotif.com

26Section D: Troubleshooting Guide
Problem/question

Recommendation

Little or no enrichment ofmethylated DNA

The Hydroxymethyl Collector kit is optimized for use with 5 ng - 2.5 µg of fragmented(<500 bp) DNA per reaction. Using different DNA concentrations will alter the amount of
b-glucosyltransferase enzyme available per DNA fragment and decrease the efficiency of
5-hmC modification. Incomplete glucosylation will reduce the recovery of 5-hmC DNA.
Make sure reagents were prepared as stated in the Buffer Preparation and Recommendation section prior to starting the assay. Reagents should be added in the order listed.

Are there alternative
incubation temperatureand times?

The glucosylation reaction is incubated at 37°C for 1 hour, but alternatively, it can be
incubated overnight at 30°C. The biotinylation reaction is incubated at 37°C for 1 hour, butalternatively, it can be incubated overnight at 30°C The streptavidin capture reaction is
incubated at RT for 1 hour, but alternatively, it can be incubated overnight at 4°C.

PCR amplification

Follow the recommendations for PCR primer design on page 22 to prepare specificprimers for your sample. The included APC PCR primers are designed to work with theincluded 5-hmC Control DNA provided in the kit.

Storage of DNA

Once DNA is enriched with Hydroxymethyl Collector, 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.

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
DNAwww.activemotif.com

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 when using enzymes that are notheat-inactivated), DNA treated in this fashion should be suitable for use in the MeDIP and
hMeDIP protocols. 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 is required for thedigest) it may be desirable to purify the digested DNA by purification columns or throughphenol extraction/ethanol precipitation.

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
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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
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