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unmethylaten DNA control


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#1 kerenshi

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Posted 25 February 2009 - 11:20 PM

hello to all!! B) :D
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 :D

#2 methylnick

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Posted 25 February 2009 - 11:37 PM

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

All comments and communication are my own personal ones, and are not tied to any of my affiliations. 


#3 kerenshi

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Posted 25 February 2009 - 11:54 PM

thank you for your quick answer!! B)
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

#4 methylnick

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Posted 01 March 2009 - 12:48 PM

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

All comments and communication are my own personal ones, and are not tied to any of my affiliations. 





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