I am master student and have DNA samples extracted from breast cancer cell lines and need to study epigenetic on them but i have quest.
So please check the following points that show DNA digestion step for subsequent use in methylation Elisa kit and as mentioned in my kit protocol i have to use sodium acetate and Tris on my DNA samples but unforunately did not verify the recommended volume
1. Extract DNA from cell or tissue samples by a desired method or commercial DNA Extraction kit.
2. Dissolve extracted DNA in water at 0.1-1 mg/mL.
3. Convert DNA sample to single-stranded DNA by incubating the sample at 95ºC for 5 minutes and then rapidly chilling on ice.
4. Digest DNA sample to nucleosides by incubating the denatured DNA with 5-10 units of nuclease P1 for 2 hrs at 37ºC in 20 mM Sodium Acetate, pH 5.2, and following with treatment of 5-10 units of alkaline phosphatase for 1 hr at 37ºC in 100 mM Tris, pH 7.5.
waiting your answers.