Problem after Bisulfite treatment

[krystaly]

Hi!

I'm new! And I have a big trouble.
I've tried 9 bisulfite treatment in 6 month, I've changed 2 methilation kit (Chemicon and Sigma), but I have no modification!!!

In a first moment I thought it's a tratment problem so I've changed time of incubation (1 h, 5.30 h, 6h, 12h,24h) temperature of incubation (55°C, 65°C, 95°C), I've tried to boiled at 100°C the DNA for 10 min before the treatment for a sure complete disassociation... I've changed every thing. but... no modification! I think it's impossible! When I do the PCR I've a good amplification but after cloning and sequencing I've no modification, my sequece is the same.

I 've tried to change 3 primers set...Now I use primer with 3 T at the end, so if the fragment is modified they could attach, otherwise no.

Please help me! I've tried everything!! This job is for my thesis and I'm desperate!

Someone have an idea?

Thanks a lot..