Is anybody currently doing bisulphite treatment or methylation specific PCR? I am doing bisulfite treatment and then methylation specific PCR, but not getting the desired result.So, I want to know whether there is problem in bisulfite treatment or in PCR. That is why, I want to know is there any way to find whether the bisulfite treatment is complete or not?
Thanks
Hi,
I think you have at least two options: bisulfite sequencing or restriction digest with enzymes which digest or not depending on bisulfite treatment. The restriction digest would be much faster and cheaper and you will get a general impression, but only for the specific recognition sequences. The sequencing approach would be the "gold standard" but if you have no access to NGS you would need to clone the BS-PCR products and you "only" get information for the specific region(s) you had amplified.
..but think also of the possibility that the obtained results are maybe not the desired ones, but the true ones :-)
Good luck.
Hi,
I think you can try to run PCR using primers for gDNA. Converted bsDNA should not give any bands with the primers constructed for gDNA.
I just tried to do it and learned that some companies provide high and low methylation control that you can use for your comparisons.
I would like to mention that its not very possible to get a know how as to whether the DNA that you are dealing with have completed the bisulfite reaction. But as Regina mentioned, its a good tactical method of determining. I also faced some problems but then I adopted the Qiagen kit for conversion and also the Qiagen Hotstar MM for the PCR. I got good results. Try that out.
Best.
Hi
It's a good way to try Angel-man primers to test your bisulfite treatment. If you had the band after using mentioned primers, then your treatment is OK.
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