4 replies to this topic

[Leon Liu]

Hello, everyone,

I'm new here and interested in DNA methylation regulation. I use the EZ methylation kit to do treatment of bisufite, and then I stored my treated DNA at -80^oC. Do someone have an idea that whether the proportion of methylation in bisulfited DNA will change with longterm conservation or frequent temperature changes (thawing for experiment)??? And  have you tested it ? (I wonder the strand which contains uracil, it maybe unstable, so it's easy to break?)

Thans a lot !

Leon

Edited by Leon Liu, 21 November 2012 - 05:51 AM.

[bob1]

The methylations shouldn't be removed, but the integrity of the DNA and hence the dectection of the methylation by bisulphite PCR may be affected by freeze/thaw.  Storage frozen at -80 should be enough to prevent degradation with time.

[Leon Liu]

bob1, on 21 November 2012 - 11:54 AM, said:

The methylations shouldn't be removed, but the integrity of the DNA and hence the dectection of the methylation by bisulphite PCR may be affected by freeze/thaw.  Storage frozen at -80 should be enough to prevent degradation with time.

Thank you for your answer, so you mean that for the bisulfited DNA, the strands which contain uracils, and others which contain cytosines (methylated), the stability for these two strands are the same???

For example, I've a bisuflted DNA which is already known that it's 50% methylated by sequencing, and then I stored this bisulfited DNA (the same one which is sequenced) at -80 for about several weeks, and I re-send this bisulfited DNA to sequence, you think it'll still be 50% methylated???

Waiting for your answers...Thank you so much!

Edited by Leon Liu, 21 November 2012 - 01:10 PM.

[bob1]

I think it should be still methylated, but I'm no expert...

[Leon Liu]

bob1, on 22 November 2012 - 12:05 PM, said:

I think it should be still methylated, but I'm no expert...

Thank you !