This product is no longer available in the United States. For a similar product, please see the MethylFlash Methylated DNA Quantification Kit (Colorimetric)





The Methylamp™ Global DNA Methylation Quantification Kit is a complete set of essential components which enables the experimenter to quantify global DNA methylation through an ELISA-like reaction using Epigentek's uniquely simplified and streamlined procedure. The entire procedure can be completed within only 4 hours in either manual or high throughput analysis. The Methylamp™ Global DNA Methylation Quantification Kit is suitable for detecting global DNA methylation status using genomic DNA isolated from variety of cultured cells, fresh and frozen tissue, paraffin-embedded tissue, plasma/serum sample, and body fluid sample, etc.

WHY CHOOSE THE METHYLAMP™ GLOBAL DNA METHYLATION QUANTIFICATION KIT?

• Very rapid procedure, which can be finished within 4 hours.
• Colorimetric quantification without radioactivity, extraction, and chromatography.
• Strip microplate format makes the assay flexible: manual or high throughput analysis.
• Simple, reliable, and consistent assay conditions.

The Methylamp™ Global DNA Methylation Quantification Kit contains all reagents required for quantification of global DNA methylation. In this assay, DNA is immobilized to the strip well specifically treated to have high affinity to the DNA. The methylated fraction of DNA can be recognized by 5-methylcytosine antibody and quantified through an ELISA-like reaction. The amount of methylated DNA is proportional to the OD intensity.

SCHEMATIC PROCEDURE:
	Quantification of Methylated DNA.

GM1 (10X Wash Buffer)
GM2 (DNA Binding Solution)
GM3 (Methylated DNA Control, 100 µg/ml)*
GM4 (Block Solution)
GM5 (Capture Antibody, 1 mg/ml)*
GM6 (Detecting Antibody, 100 µg/ml)*
GM7 (Developing Solution)
GM8 (Stop Solution)
8-Well Assay Strips (with frame)
User Guide

* Spin the solution down to the bottom before use.

1. OD for blank is high, what are some suggestions to correct this?
Increase washes, extend block time to 1 hour with GU4, or decrease color development time. The stop solution should be added when the color in the positive control wells have changed to brilliant blue, and the color in blank wells have not changed, or may have only changed to slight blue. The OD for blank in general is 0.15-0.3, while OD for the positive control at 20 ng is 1-1.2. Also, increasing dilution of GU7 will help reduce background noise.

2. What is the sensitivity and DNA binding capacity of this kit?
Sensitivity limitation of the methylated DNA detection is about 0.2 ng. The reproducibility is quite good based on our quality control and customer feedback. CV% is 5-10% between wells, and 10-15% between strips, and 15-25% between different tests. The binding capacity of the well is about 400 ng of DNA.

3. Should DNA be sheared (or treated with an enzyme) prior to binding to wells?
Both un-fragmented and fragmented DNA can be tightly bound to the assay wells.

4. What is the antibody specificity of this kit?
The antibody used in the kit is monoclonal anti-5-methylcytosine Ab which recognizes any methylcytosine including 5-meC and 5-medC in CpG sites or non-cpG sites.

5. Can the kit be used for Human DNA/any model organism?
Yes.

6. Can sample DNA be frozen before testing?
Yes.

7. Is it necessary to know the GC content to use this assay?
No, the global DNA methylation can be quantified by comparing the amount of measured methylated DNA fraction to total DNA added for assay.

8. What are the minimum DNA quantities required for this assay?
Based on our testing, the DNA amount can be as low as 25 ng, but 50 ng DNA per each assay point is recommended.

9. Should the well be covered when incubated at 37°C for 2 hours?
No, the purpose is to dry the wells.

10. After adding stop solution, there were black precipitates in the wells.
Too strong of a reaction occurred; decrease the color development time to reduce the color reaction.

11. Kit results were fine, but OD values for samples were negative.
Increase amount of sample DNA.

12. Result for negative control gives a positive OD and there is no difference in OD between different concentrations of the positive control.
The low OD value for the positive control could be due to: 1) no binding of control DNA to the substrate coated on the strip well, and 2) capture antibody is less active. Please be sure to check the appropriate temperature and storage of the antibody and strip wells. (Capture antibody and 8 well assay strips should be stored at 4°C. Detecting antibody should be stored at -20°C.)

13. Can you explain the low OD values of my target DNA?
The meDNA fraction generally accounts for 0.8-1.5% of total DNA. Increased methylation of DNA would have higher fraction of meDNA (>2%). 200 ng of genomic DNA could contain 1.5-3 ng of meDNA and normally most of the DNA samples at this amount would give a low OD.

[User Guide]* *Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Material Safety Data Sheet]

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