When Methylation Results are not Reproducible - (Aug/25/2007 )
I successfully bisulphite converted and sequenced my promoter region using Zymo kit. After a week's gap and two weeks of frustration, I am not able to reproduce the results. I see no PCR amplification product. Instead i see non-specific bands. These band sizes also appear with un-bisulphite treated DNA.
When you see results like this, does it mean
1. the DNA starting material was not good quality
2. the bisulphite in the kit was poor quality
3. the primers are not well-desgined, so sometimes they work v.well and other times they do not.
4. any thing else ? i should take care of?
Any help will be greatly appreciated.
I'd guess it is the quality or starting amount of your template DNA. Maybe there is a problem with the charge of the kit, you can try out a new one. Primers should either work or not. Another possibility is a problem with the cycler (like not heating anymore etc.)