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Positive control and Negative control - (Sep/05/2007 )

Could anyone provide information where to buy positive control and negative control for DNA methylation in bisulfite-sequencing? Thanks!

-sallynie-

hi sallynie, when you mean controls, do you mean hypermethylated and hypomethylated DNA for bisulfite conversion?

or do you mean, DNA that is known to be bisulfite converted and DNA that is not?

Nick

-methylnick-

Yes, I think so! I know people frequently use Methyltransferase treatment genome DNA as positive control. Right? But what is the negative control? My supervisor suggest me to buy them if they are available. Do you have some suggestion for them?

-sallynie-

New England Biolabs have a set of methylation (SssI methylase) and un-methylated (5-aza-dC) human DNA controls for sale. Haven't used them just yet, but will do so in the very near future.

For unmethylated DNA we just take genomic DNA and whole genome amplify 100ng with Sigma's WGA kit which gives you effectively genomic DNA with no methylation and it seems to work rather well.

Good luck!

Nick

-methylnick-

The universally methylated and unmethylated DNA by NEB is very good. For calibrating measurements it is my impression that it is more reliable than doing the sss1 treatment yourself.
Kr├╝mel

-kr├╝melmonster-

We use a negative control from Chemicon...CpGenome Universal Unmethylated DNA....it comes in two vials. Vial A contains genomic DNA and Vial B contains genomic DNA isolated from a primary foetal cell line. You're advised to test both unmethylated controls out. For example, i found the vial B DNA was actually methylated in some areas of my gene and not others.

-imprinter-