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bisulfite treatment - in my hands bisulfite kit don't work well (Jan/22/2007 )

hi..
, i use 2 kit for bisulfite treatment and they are: EZ kit and qiagen kit
but I have a lot of problems...
when i use qiagen kit i have not amplicone but when i use EZ kit...i have amplicon but i have other problems...

i treat my genomic DNA and then I make a PCR using primers that have not modified...i have the amplicon and i clone in TOPOTA and then sequencing...
but when i analize the sequence of my gene promoter i see only few C convert in T...
and i see that cytosine also near other C, or near T and A don't convert...
how is it possible?
maybe my kits don't work well or my primers amplified only genomic DNA that is not modified completely...
have you observed this thing?

bye bye sad.gif

-full-methy-

hi full-methyl,

I have been there before,

you said you used primers that were to unmodified DNA, this is the reason why you are seeing very low conversion rates.

Have a look at my pinned thread with regard to primer design.

Nick

-methylnick-