3 replies to this topic

[kerenshi]

hello to all!!   
I am looking for unmethylated DNA control. the methylation status of my gene is unknown and thus I can't use PBL or other normal tissue. I found the unmethylated DNA control of Millipore and also the unmethylated DNA control of QIAGEN (Epitec catalog No. 59665) . both are expensive. did any one use those controls succesfully? what about using salmon sperm instead? I understand that salmon sperm DNA is unmethylated. can I use it instead? did anyone used it? protocol and tips are welcome!!!

thanks

[methylnick]

salmon sperm DNA for methylation is great only if you are studying salmon!!!!!

The millipore methylated DNA works well, the unmethylated one would depend on your gene of interest.

I know they are expensive, you may be better off to make your own.

Nick

[kerenshi]

thank you for your quick answer!!
slamon -hhhhh offcourse!

O.K - what do you mean when you wrote that the unmeth DNA is depend on my gene? isn't all DNA is unmeth?
and
how do I make my own unmethylated DNA without cloning the genomic region ? is DNA from cells treated with 5-AZA is good enough to be a control?
do you have protocol to generate them?
thank you
keren

[methylnick]

well, the unmethylated control is just from a human fetal cell line and we have found that there was methylation albeit residual in this sample bought from Millipore.

The way to make your own is to use a PCR/ampification based method to generate your DNA, something like whole genome amplification whereby the methylation mark is erased upon amplification is suitable, I just WGA2 from sigma following protocols, it works a treat.

nick