SSSI positive Control degrading? - (Jun/07/2007 )
Hi everyone,
I've been routinely methylating gDNA (from my blood) with NEB's SSSI kit according to their protocol to use as positive control in MSP. The controls work fine for a week, but soon after, the bands disappear. I can't come up with a reason for this as all my other bisulfite treated DNA (cell line DNA / blood DNA but unmethylated) stays stable for weeks at-20°C, enduring multiple freeze thaw cycles.
Heres the procedure:
Isolate DNA from blood / cell line: DNeasy, Qiagen
Methylation: SSSI, NEB
BS mod.: Epitect, Qiagen
Have any of you had similar experience? Any hints? Or should I just keep on making fresh control each week (time waste galore...)
cheers
Cyburn
That would at least explain my latest difficults ...
cyburn, do you inactivate your SssI methylase reaction?
Nick
Nick
Only with a step in the program: 65°C for 20 mins. Is additional treatment required?
i inactivate the reaction and also column purify the DNA because you may have residual activity of SssI sos I would remove it completely.
Nick
Nick
yeah, I run it all through a Promega Wizard CleanUp Column after the methylation program is finished (should've mentioned that
) So your SSSI controls are more durable? hmm yeah....
so do you elute in water or TE? I don't think this should make a difference, but we elute in TE.
Nick
so do you elute in water or TE? I don't think this should make a difference, but we elute in TE.
Nick
Well I elute in Water but then immediately BS modify, that I do elute in TE (Qiagen EB Buffer). The other DNA is also eluted in water. It's all rather puzzling...
Hi cyburn, you directly do the BS modification? Than there should be no degradation of the methylation as it is fixed in the sequence
BS modified DNA should be stable at least for some weeks...
BS modified DNA should be stable at least for some weeks...
exactly, that's why I'm so puzzled! I can't make any sense of it at all. Maybe some kind of degrading agent is introduced by either the SSSI reaction or the Promega Wizard Cleanup kit, as those are the 2 steps that the other samples aren't exposed to. But that seems pretty farfetched, too.
I am not able to amplify from the positive controls at all after some time. Neither MSP nor BSP.