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Checking my own Methylated Control for MSP

SssI HpaII

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#1 Bianabi

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Posted 21 October 2011 - 03:36 AM

I’m making my own Methylated Control after having some contradictory results with Epitect Controls DNAs from QIAGEN.
I’ve treated human DNA (from PBMCs) with M.SssI from Zymo (30ºC overnight and then re-addiction of enzyme 4 more hours) and purified according to a standard Phenol:Chlorophorm protocol.
I’m not able to sequence it yet in order to check if it’s really completely methylated so I’ve digested 200ng of converted DNA with 4 Units of HpaII and another 200ng with MspI (37ºc overnight).
I don’t know how to interpret the result of my 0.8% agarose gel. I see a range of “background” in both cases. The only difference is that MspI well has run faster than HpaII and it’s more intensive.
I expected to see a big high band in HpaII and different bands in MspI but I’m not sure if this happens with genomic DNA (I’ve seen some of you perform this with plasmids).
May 200ng be too low quantity?
I really need help because my MSPs have been stopped such a long time waiting for a control!
Thank!

#2 methylnick

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Posted 26 October 2011 - 07:46 PM

sounds like your M.SssI hasn't worked efficiently. you may want to try because it's genomic DNA a couple of rounds of methylation using this enzyme, or even spiking it with more SAM as it depletes.

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