ab156901 –
Universal Methylated
DNA Preparation Kit
Instructions for Use
For the generation and purification of DNA methylated at CpG sites
This product is for research use only and is not intended for diagnostic use.
Version 1 Last Updated 3 September 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2
2.
ASSAY SUMMARY
3
GENERAL INFORMATION
3.
PRECAUTIONS
4
4.
STORAGE AND STABILITY
4
5.
MATERIALS SUPPLIED
5
6.
MATERIALS REQUIRED, NOT SUPPLIED
5
7.
LIMITATIONS
6
8.
TECHNICAL HINTS
6
ASSAY PREPARATION
9.
REAGENT PREPARATION
7
10.
SAMPLE PREPARATION
7
ASSAY PROCEDURE
11.
ASSAY PROCEDURE
8
RESOURCES
12.
NOTES
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1
INTRODUCTION
1. BACKGROUND
Epigenetic inactivation of genes plays a critical role in many important
human diseases, especially in cancer. A core mechanism for epigenetic
inactivation of the genes is methylation of CpG islands in genome DNA.
Methylation of CpG islands involves the course in which DNA
methyltransferases (Dnmts) transfer a methyl group from S-adenosyl-Lmethionine to the fifth carbon position of the cytosines. Aberrant DNA
methylation is mainly found in 5’-CpG-3’dinucleotides within promoters or in
the first exon of genes, which is an important pathway for the repression of
gene transcription in diseased cells. It is well demonstrated that DNA
methylation plays an important role in the regulation of gene expression,
tumorigenesis, and other genetic and epigenetic diseases; thus, detection of
methylation in some genes of diseased cells could provide very useful
information for discrimination of that disease. There have been many
methods such as methylation-specific PCR (MS-PCR) for the detection of
DNA methylation. A methylation-positive control could be required for
successful performance of gene methylation studies.
Abcam’s Universal Methylated DNA Preparation Kit (ab156901) provides a
tool for generating methylated DNA at CpG sites as the methylation-positive
control used for methylation studies. The kit includes all the components
necessary for generating and purifying methylated DNA. The kit is sufficient
for methylating 40 µg of DNA. This kit is suitable to be used with Abcam’s
DNA Modification Kit series. It can also be used with other DNA modification
or methylation kits.
This kit allows the generation of methylated DNA using DNA from various
sources including genomic DNA, plasmid DNA and oligonucleotides.
Double-stranded DNA should be used with this kit. DNA size can be from
40 bp to full length of genome; DNA quantity can be from 500 ng to 40 µg,
optimal at 10 µg. Recovery rate of methylated DNA is greater than 80%.
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INTRODUCTION
2. ASSAY SUMMARY
Prepare DNA solution
Add DNA solution to methylation reaction mix and incubate at 37°C
Add purification buffer and 100% ethanol and incubate at -20°C
Centrifuge, remove supernatant and wash with 90% ethanol
Repeat wash with 90% ethanol
Dry DNA pellet at room temperature
Add elution buffer to dissolve methylated DNA
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3
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit as given in the table and away from light upon receipt.
Observe the storage conditions for individual prepared components in
sections 9 & 10.
For maximum recovery of the products, centrifuge the original vial prior to
opening the cap.
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GENERAL INFORMATION
5. MATERIALS SUPPLIED
4 mL
Storage
Condition
(Before
Preparation)
RT
Methylation Buffer
400 µL
–20°C
Adomet, 30 mM
50 µL
–20°C
Methylation Mix
26 µL
–20°C
Purification Buffer
4 mL
RT
Elution Buffer
1 mL
RT
Item
Reaction Buffer
Quantity
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:




Pipette
100% and 90% Ethanol
Incubator or water bath for 65°C incubation
Microcentrifuge capable of 12000 rpm
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GENERAL INFORMATION
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures

Do not use kit or components if it has exceeded the expiration date on
the kit labels

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and performance
cannot be guaranteed if utilized separately or substituted

Any variation in operator, pipetting technique, washing technique,
incubation time or temperature, and kit age can cause variation in
binding
8. TECHNICAL HINTS

Avoid foaming or bubbles when mixing or reconstituting components

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions

Ensure plates are properly sealed or covered during incubation steps

Complete removal of all solutions and buffers during wash steps

This kit is sold based on number of tests. A ‘test’ simply refers to a
single assay well. The number of wells that contain sample, control
or standard will vary by product. Review the protocol completely to
confirm this kit meets your requirements. Please contact our
Technical Support staff with any questions
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ASSAY PREPARATION
9. REAGENT PREPARATION
All reagents provided are ready to use.
10. SAMPLE PREPARATION
DNA: Dilute DNA to 500 ng/µL with DNAse/RNAse free water. DNA size
can be from 40 bp to full length of genome; DNA quantity can be from
500 ng to 40 µg, optimal at 10 µg.
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ASSAY PROCEDURE
11. ASSAY PROCEDURE
11.1 Prepare the reaction mix (for 10 µg of DNA) in a 1.5 mL vial:
Component
DNA Solution (500
ng/µL)
Reaction Buffer
Methylation Buffer
Adomet
Methylation Mix
Quantity (µL)
20
61
10
2.6
6 (finally added)
11.2 Mix well by pipetting 5-6 times. If DNA concentration is less than
500 ng/µL, you can increase the volume of the DNA Solution (up to
80 µL) and reduce the volume of the Reaction Buffer.
11.3 Tightly cap the vial and incubate at 37°C (waterbath or thermal cycler)
over night.
11.4 Add 100 µL of the Purification Buffer and 800 µL of 100% ethanol into
the vial. Incubate at -20°C for 1-2 hours.
11.5 Centrifuge at 12,000 rpm for 10 minutes. (At this step, you may see
precipitates at the bottom of the vial).
11.6 Carefully remove the supernatant. Then add 500 µL of 90% ethanol
into the vial, and centrifuge at 12,000 rpm for 30 seconds.
11.7 Carefully remove the supernatant. Add 500 µL of 90% ethanol again
into the vial, and centrifuge at 12,000 rpm for 35 seconds. Carefully
remove the supernatant as much as possible.
11.8 Leave the vial open for 10-15 minutes at room temperature to dry.
Add 60-70 µL of Elution Buffer into the vial. Pipette the solution
several times to dissolve methylated DNA.
11.9 Store DNA at -20°C (for up to 6 months) or use immediately
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RESOURCES
12. NOTES
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RESOURCES
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