Sample Pretreatment Procedure for 8-OHdG Detection.
To assay properly, please pre-treat samples desired below. In addition, please avoid repeated freeze thaw samples. In order to confirm the aptitude of assay methods on a new sample, implementing recovery test of standard 8-OHdG added into the new sample is recommended.
If it's clear, pretreatment is not needed. Centrifugation at 2,000~5,000g for 10 ~15 minutes is recommended for opaque samples only.
Blood samples must be separated to serum immediately. To separate interfering substances, filtration of serum using an ultra filter (cut off molecular weight 10,000) is necessary.
Prepare ultra-filter following manufacture's protocols.
In order to reduce deviation, diluting samples by more than twice, while paying attention to concentration range is suggested.
DNA Sample from organs and cultured cells:
It is necessary to extract samples and digest DNA before using this kit.
1. Extract DNA from cells.
Dextract DNA from the homogenates using a DNA extraction kit. Please follow the manufacture's manual.
2. Calculation of DNA Concentration and Purity.
a) DNA Solution is diluted to 20-50 micro g/mL with TE buffer.
b) Measure absorbance at wavelength 230nm, 260nm, 280nm and 320nm.
c) Calculate DNA Concentration. (1.0 of Absorbance at 260nm is equal to 50 micro g/mL DNA)
DNA concentration (micro g/mL) = (Absorbanve at 260nm) * (50 micro g/mL) * dilution rate
d) Check the purity of DNA sample.
The ratio of (Absorbance at 260nm) : (Absorbance at 280nm) is typically between 1.8 to 1.85.
The purity of isolated DNA is assessed by the ratio of (Absorbance at 260nm) : (Absorbance at 230nm), and should be between 2.2 to 2.25.
3. Enzymatic Digestion of DNA.
a) Dissolve 200 micro gram of extracted DNA in 135 micro L of water.
b) Add 15 micro L of 200mM sodium acetate and 6 units of nuclease P1 (15 micro Ll, 1 mg/mL) to the DNA Solution, and then incubated for 30 min to 1hr at 37 degree C after Argon substitution.
c) Add 1M Tris-HCl buffer (15 micro L, pH 7.4) and 2 unit of alkaline phosphatase (7 micro L, 200 units/0.7mL), and incubated for 30 min to 1hr at 37 degree C after Argon substitution.
d) To remove enzymes and other macromolecules, the hydrolysate are filtered through Millipore Ultra free C3LGC at 14000rpm for 10min.
e) Apply 50 micro L of DNA digest to the wells of ELISA kit. It is recommended that analysis procedure mentioned here shoukd be completed in one day.
a) Water: Purified Water.
b) TE buffer: 1mM EDTA in 10mM Tris-HCl(pH8.0)