关键词：DNA甲基化 重亚硫酸盐 DNA测序
DNA methylation ；bisulfite；DNA sequencing
1. Digest the target DNA with restriction enzyme that doest not cut within the sequence of interest, or shear the DNA by passing through a narrow gauge needle. (This step is important for genomic DNA). Typically, 50ng to 2μg DNA could be used for conversion (usually in 50μl). Ensure that DNA is free of residual protein and RNA.
2. Recover the DNA. Elute with 90μl(usually add 95μl into the minicolumn to collect 90μl DNA solution).
3. Denature the DNA by the addition of 10μl fresh 3M NaOH to final concentration of 0.3M, incubate at 37~42℃ for 15~20min.
4. Prepare a fresh solution of (saturated) 2.3M sodium metabisulfite, pH5.0(see the end of the protocol), for full solution of the metabisulfite(Do not vortex). Add 1040μlbisulfite solution and 30μl 20mM hydroquinone to DNA and supplement with 20μl water, mix by gently inverting, overlay with mineral oil, and incubate at 55℃ for 8~16hr (or overnight)
5. Recover the DNA from underneath the oil, and desalting by a column. Elute the DNA with 90μl water(95μl to the column).
6. Desulfinate by adding 10μl 3M NaOH (final concentration is 0.3M), and incubate in 37℃ for 15min.
7. Neutralize by adding 70μl ammonium acetate to pH7.0 and precipitate by adding 400μl ethanol(usually 3 volumes of the solution). Adding 10-20μl glycogen to facilitate precipitation. Resuspend DNA in 10μl water or store at -20℃ until use.
Preparation of metabisulfite solution:
4ml: 1.76g sodium metabisulfite(Sigma)+340μl3M NaOH+3ml H2O
Mix gently (Don’t vortex)
Notice: You should prepare fresh solution (2.3M sodium metabisulfite, 20mM hydroquinone) on the day you want to treat DNA!!!
尽信书则不如无书，里面的具体操作也许跟贵实验室的一些具体条件不相匹配，还望能根据实验的原理灵活把握。
有什么不清楚的地方，敬请批评指正。
祝好运。
