Q: Can this be used on samples from any species?
A: The Global DNA Methylation ELISA kit is not species specific because the 5MedCyd modification is the same for all species.
Q: Can I use this on DNA isolated from formalin fixed paraffin embedded tissues?
A: This kit can be used on any sample as long as DNA can be extracted. It is therefore fine to use on DNA isolated from formalin fixed paraffin embedded tissues.
Q: Is this kit suitable for use with bacterial cells?
A: Since this kit uses isolated DNA samples, it is suitable for use with DNA extracted from bacteria.
Q: What is the minimum amount of DNA that can be used with this kit?
A: A minimum of 2 µg DNA is required for this assay.
Q: Is digestion with nuclease P1 necessary?
A: Yes, DNA digestion is important with this assay because the DNA is recognized by the antibody as single nucleotides. Untreated samples or samples that have incomplete digestion will not be recognized and the results will be unreliable.
Q: Do you have a recommended nuclease P1 and alkaline phosphatase?
A: We generally recommend nuclease P1 from Sigma #N8630 and alkaline phosphatase from Sigma #P5931, but any commercially available reagents are fine.
Q: Can digested DNA be stored before testing?
A: The digested DNA is stable and can be stored at -20ºC for up to six months.
Q: Do you have a sample preparation protocol?
A: You may use the following example calculation as a guideline:
1. Extract DNA from cell or tissue samples by a desired method or commercial DNA Extraction kit. Dissolve extracted DNA in water at 1 mg/mL.
2. Add 135 µL of DNA sample in a tube and convert DNA sample to single-stranded DNA by incubating the sample at 95ºC for 5 minutes and rapidly chilling on ice.
3. Add 15 µL of 200 mM Sodium Acetate, pH 5.2 and 5 units of nuclease P1 (e.g. Sigma N8630, reconstituted in 30 mM Sodium Acetate, pH 5.2 and 5 mM Zinc Chloride) to the denatured DNA sample and incubate for 2 hrs at 37ºC.
4. Add 15 µL of 1M Tris, pH 7.5, and 5 units of alkaline phosphatase (e.g. Sigma P5931, reconstituted in 1 mM Magnesium Chloride), and incubate for 1 hr at 37ºC.
5. The reaction mixture is centrifuged for 5 minutes at 6000 g and the supernatant is used for the 5MedCyd ELISA assay or stored at -20ºC for up to 6 months.
Note: When 50 µL of the nuclease digestion mixture is used in the kit, about 40 µg DNA is used.
Q: How do I make the standard curve and calculate my results?
A: The best way to determine concentrations from a standard curve is to use a 4-parameter curve fitting program, but if you don’t have this software it can be done using Excel. When graphing the standard curve, set the x-axis to logarithmic scale and generate a linear or logarithmic trendline. The standard curves generated with ELISAs are not typically linear, but a linear curve can be created by eliminating the upper and lower values of the curve, as long as the sample values fall within this range. The middle part of the standard curve is the most sensitive and is the best part to use for quantifying samples. The equation of the trendline can be used to calculate the concentrations of unknowns by solving for x in y=mx+b, which is (OD value-b)/m for linear lines or ln(x)=(OD value-b)/m for logarithmic lines.