我的预想实验步骤大致如下：
DNA的bisulfite处理；甲基化PCR; 变性聚丙酰胺凝胶电泳； 377或3100 测序（非310或3700；非毛细管型）
在《Current protocols of Human Genetics》里面也有《Methylation-specific PCR》这一章，列出了需要的材料：
Materials
Sample DNA
Positive control DNA, previously determined to be methylated
Negative control DNA, previously determined to be unmethylated
2 M and 3 M NaOH
10 mM hydroquinone (prepare fresh before use; see recipe)
3 M sodium bisulfite (prepare fresh before use; see recipe)
Mineral oil
DNA Wizard cleanup kit (Promega) or equivalent
80% isopropanol, room temperature
Autoclaved distilled water, 60° to 70°C and room temperature
Electrophoresis buffer (preferably TBE buffer; APPENDIX 2D)
10 mg/ml glycogen
10 M ammonium acetate
100% and 70% ethanol, ice cold
10× PCR amplification buffer (APPENDIX 2D) with 15 mM MgCl2
25 mM 4dNTP mix (APPENDIX 2D)
300 ng/µl sense and antisense primers
Taq DNA polymerase
Vacuum manifold
Dedicated micropipettors for setting up PCR reactions
0.5-µl PCR tubes or strips
Thermal cycler with tube-controlled temperature monitoring
Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis and gel staining and photography作者: tie8 时间: 2016-1-7 17:14
比较了肿瘤所的秘方和这些protocol，大致相同，在部分试剂和DNA的量方面有些区别（这可能是经验吧）。
1.看来《Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands （1996）》这篇文章，有些初级疑问：里面加的1ug samon sperm DNA (SIGMA) 是做载体的吧？
2.在《Methylation-specific PCR》里面，必需的材料也包括Positive control DNA, previously determined to be methylated和Negative control DNA, previously determined to be unmethylated，这如何办？
3.有用过3100或377在这方面的经验吗？两者有何区别？
附件论文：Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands （1996）太大了，1.6M， 如有所需，PM。作者: tie8 时间: 2016-1-7 17:17
图片附件: 93046945.jpg (2016-1-7 17:14, 37.04 KB) / 该附件被下载次数 22
